Compositions and Methods for Preventing and Treating Salmonella Typhi and Salmonella Paratyphi Infection

ABSTRACT

The invention provides compositions and methods for preventing, treating and diagnosing infection by  Salmonella enterica  serovar  typhi  ( S. typhi ) and/or  S. paratyphi,  i.e., typhoid fever.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationSer. No. 61/825,375, filed May 20, 2013, which is hereby incorporated byreference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberAI079022 awarded by the National Institute of Allergy and InfectiousDiseases. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Salmonella enterica serovar Typhi (S. typhi) and paratyphi (S.paratyphi), the causes of typhoid fever, results in more than 200,000annual deaths (Parry et al., N. Engl. J. Med. 347:1770-1782; Crump etal., 2008, Antimicrob. Agents Chemother. 52:1278-1284; Raffatellu etal., 2008, J. Infect. Dev. Ctries. 2:260-266; Butler, 2011, Clin.Microbiol. Infect. 17:959-963; Crump and Mintz, 2010, Clin. Infect. Dis.50:241-246). Unlike other Salmonella serovars, which typically causeself-limiting gastroenteritis, S. typhi and S. paratyphi cause asystemic, life-threatening disease (Parry et al., N. Engl. J. Med.347:1770-1782). The genomes of S. typhi and S. paratyphi contain adearth of unique virulence factors that are not found in non-typhoidalserovars, and the molecular bases for its unique virulence propertiesand clinical presentation are unknown (Sabbagh et al., 2010, FEMSMicrobiol. Lett. 305:1-13; Parkhill et al., 2001, Nature 413:848-852).

One of the few S. typhi- and S. paratyphi-specific factors that has beenshown to directly impact its interaction with host cells is an AB-typetoxin dubbed typhoid toxin (Haghjoo and Galan, 2004, Proc. Natl. Acad.Sci. USA 101:4614-4619; Spano et al., 2008, Cell Host Microbe 3:30-38;Spano and Galan, 2008, Curr. Opin. Microbiol. 11:15-20). AB familytoxins consists of enzymatically active A subunits that interfere withhost functions, and B subunits that deliver the toxins to their targetcells through receptor-mediated endocytosis (Beddoe et al., 2010, TrendsBiochem. Sci. 35:411-418; Merritt and Hol, 1995, Curr. Opin. Struct.Biol. 5:165-171). Unlike typical AB toxins, typhoid toxin is composed oftwo A subunits, PltA and CdtB, which are homologs of the A subunits ofthe pertussis and cytolethal distending toxins, respectively

(Spano et al., 2008, Cell Host Microbe 3:30-38). Its single B subunit,PltB, is a homolog of one of the components of the heteropentameric Bsubunit of pertussis toxin. Although the cellular targets of theADP-ribosyl transferase activity of P1tA have not yet been identified,CdtB is a deoxyribonuclease that is targeted to the nucleus where itinflicts DNA-damage and induces cell cycle arrest (Haghjoo and Galan,2004, Proc. Natl. Acad. Sci. USA 101:4614-4619; Lara-Tejero and Galan,2000, Science 290:354-357; Lara-Tejero and Galan, 2002, Trends inMicrobiology 10:147-152).

This toxin is remarkable in that the activities of two powerful toxinsseem to have been co-opted into a single toxin with unique biology.There are currently no effective vaccines to protect against typhoidfever, and in particular to protect young children, the most susceptiblepopulation, against typhoid fever. Moreover, there are currently noeffective and specific diagnostic tools for typhoid fever, and multipleantibiotic resistant S. Typhi is rapidly emerging, with the prospects oftyphoid fever being untreatable by antibiotics becoming a real threat(Butler, 2011, Clin. Microbiol. Infect. 17:959-963). Thus, there is aneed in the art for compositions and methods for preventing, treatingand diagnosing typhoid fever. The present invention addresses this unmetneed.

SUMMARY

The invention described herein provides compositions and methods forpreventing, treating and diagnosing infection by Salmonella entericaserovar typhi (S. typhi) and/or S. paratyphi, i.e., typhoid fever. Inone embodiment, the invention is a vaccine having at least onepolypeptide selected from the group consisting of PltA, or a PltAmutant, PltB, or a PltB mutant, and CdtB or a CdtB mutant. In someembodiments, the PltA mutant is PltA E133X, relative to SEQ ID NO: 8. Insome embodiments, the PltA mutant is PltA E133A, relative to SEQ ID NO:8. In some embodiments, the PltB mutant is PltB S35X, relative to SEQ IDNO: 9. In some embodiments, the PltB mutant is PltB S35A, relative toSEQ ID NO: 9. In some embodiments, the CdtB mutant is CdtB H160X,relative to SEQ ID NO: 7. In some embodiments, the CdtB mutant is CdtBH160Q, relative to SEQ ID NO: 7. In some embodiments, the CdtB mutant isCdtB R119X, relative to SEQ ID NO: 7. In some embodiments, the CdtBmutant is CdtB H259X, relative to SEQ ID NO: 7. In some embodiments, theCdtB mutant is CdtB ΔCys269, relative to SEQ ID NO: 7. In someembodiments, the CdtB mutant is CdtB C269X, relative to SEQ ID NO: 7. Insome embodiments, the PltA mutant comprises any mutation in PltA thatdisrupts its enzymatic activity. In some embodiments, the CdtB mutantcomprises any mutation in CdtB that disrupts its enzymatic activity.

In another embodiment, the invention is a method of immunizing a subjectagainst S. typhi, the method including the step of administering to thesubject a vaccine comprising at least a portion of at least onepolypeptide selected from the group consisting of PltA, a PltA mutant,PltB, a PltB mutant, CdtB, a CdtB mutant, PltA E133X, relative to SEQ IDNO: 8, PltA E133A, relative to SEQ ID NO: 8, PltB S35X, relative to SEQID NO: 9, PltB S35A, relative to SEQ ID NO: 9, CdtB H160X, relative toSEQ ID NO: 7, CdtB H160Q, relative to SEQ ID NO: 7, CdtB R119X, relativeto SEQ ID NO: 7, CdtB H259X, relative to SEQ ID NO: 7, CdtB ACys269,relative to SEQ ID NO: 7, and CdtB C269X, relative to SEQ ID NO: 7. Insome embodiments, the subject is currently infected with S. typhi or S.paratyphi and the vaccine induces an immune response against S. typhi orS. paratyphi. In other embodiments, the subject is not currentlyinfected with S. typhi or S. paratyphi and the vaccine induces an immuneresponse against S. typhi or S. paratyphi.

In one embodiment, the invention is a method of treating a subjectinfected with S. typhi, the method including the step of administeringto the subject a vaccine comprising at least a portion of at least onepolypeptide selected from the group consisting of PltA, a PltA mutant,PltB, a PltB mutant, CdtB, a CdtB mutant, PltA E133X, relative to SEQ IDNO: 8, PltA E133A, relative to SEQ ID NO: 8, PltB S35X, relative to SEQID NO: 9, PltB S35A, relative to SEQ ID NO: 9, CdtB H160X, relative toSEQ ID NO: 7, CdtB H160Q, relative to SEQ ID NO: 7, CdtB R119X, relativeto SEQ ID NO: 7, CdtB H259X, relative to SEQ ID NO: 7, CdtB ACys269,relative to SEQ ID NO: 7, and CdtB C269X, relative to SEQ ID NO: 7. Insome embodiments, the subject is currently infected with S. typhi or S.paratyphi and the vaccine induces an immune response against S. typhi orS. paratyphi. In other embodiments, the subject is not currentlyinfected with S. typhi or S. paratyphi and the vaccine induces an immuneresponse against S. typhi or S. paratyphi. In some embodiments, themethod of treating further comprises the step of administering anantibiotic to the subject.

In one embodiment, the invention is an isolated polypeptide selectedfrom the group consisting of PltA E133X, PltA E133A, PltB S35X, PltBS35A, CdtB H160X, CdtB H160Q, CdtB R119X, CdtB H259X, CdtB ACys269, andCdtB C269X. In another embodiment, the invention is an amino acidsequence selected from the group consisting of PltA E133X, PltA E133A,PltB S35X, PltB S35A, CdtB H160X, CdtB H160Q, CdtB R119X, CdtB H259X,CdtB ACys269, and CdtB C269X. In one embodiment, the invention is anantibody the specifically binds to at least one selected from the groupconsisting of PltA E133X, PltA E133A, PltB S35X, PltB S35A, CdtB H160X,CdtB H160Q, CdtB R119X, CdtB H259X, CdtB ACys269, and CdtB C269X.

In another embodiment, the invention is a method of treating a subjectinfected with S. typhi, the method including the step of administeringto the subject at least one antibody, wherein the at least one antibodyspecifically binds to at least one of PltA, PltA E133X, PltA E133A,PltB, PltB S35X, PltB S35A, CdtB, CdtB H160X, H160Q, CdtB R119X, CdtBH259X, CdtB C269X and CdtB ACys269. In some embodiments, the method oftreating further comprises the step of administering an antibiotic tothe subject.

In one embodiment, the invention is an inhibitor composition useful fortreating or preventing S. typhi infection, wherein the inhibitorcomposition inhibits the interaction between the S. typhi toxin and theS. typhi toxin receptor. In one embodiment, the inhibitor compositionspecifically binds to the S. typhi toxin. In another embodiment, theinhibitor composition specifically binds to the S. typhi toxin receptor.In one embodiment, the S. typhi toxin receptor is a glycan. In variousembodiments, the inhibitor composition is at least one selected from thegroup consisting of a chemical compound, a protein, a peptide, apeptidomemetic, an antibody, a ribozyme, a small molecule chemicalcompound, a glycan, an antisense nucleic acid molecule. In variousembodiments, the inhibitor composition comprises at least one glycanlisted in FIG. 20, 21 or 22. In some embodiments, the glycan is soluble.In another embodiment, the invention is a method of treating a subjectinfected with S. typhi, the method comprising administering to thesubject an inhibitor composition. In some embodiments, the method oftreating further comprises the step of administering an antibiotic tothe subject.

In another embodiment, the invention is a method of diagnosing an S.typhi or S. paratyphi infection in a subject in need thereof, the methodincluding the steps of determining the level of at least one of PltA,PltB, CdtB in a biological sample of the subject, comparing the level ofthe at least one of PltA, PltB, CdtB in the biological sample with levelin a comparator control, and diagnosing the subject with an infection byS. typhi or S. paratyphi when the level of the at least one of PltA,PltB, CdtB in the biological sample is elevated when compared with thelevel in the comparator control. In some embodiments, the level of theat least one of PltA, PltB, CdtB in the biological sample is determinedby measuring the level of at least one of PltA mRNA, PltB mRNA, CdtBmRNA in the biological sample. In other embodiments, the level of the atleast one of PltA, PltB, CdtB in the biological sample is determined bymeasuring the level of at least one of PltA polypeptide, PltBpolypeptide, CdtB polypeptide in the biological sample. In someembodiments, the comparator control is at least one selected from thegroup consisting of: a positive control, a negative control, ahistorical control, a historical norm, or the level of a referencemolecule in the biological sample. In some embodiments, the methodfurther comprises the step of administering a therapy to the subject totreat the infection.

In another embodiment, the invention is a method of diagnosing an S.typhi or S. paratyphi infection in a subject in need thereof, the methodincluding the steps of determining the level of antibody thatspecifically binds to at least one of PltA, PltB, CdtB in a biologicalsample of the subject, comparing the level of antibody that specificallybinds to the at least one of PltA, PltB, CdtB in the biological samplewith level in a comparator control, and diagnosing the subject with aninfection by S. typhi or S. paratyphi when the level of the antibodythat specifically binds to at least one of PltA, PltB, CdtB in thebiological sample is elevated when compared with the level in thecomparator control. In some embodiments, the comparator control is atleast one selected from the group consisting of: a positive control, anegative control, a historical control, a historical norm, or the levelof a reference molecule in the biological sample. In some embodiments,the method further comprises the step of administering a therapy to thesubject to treat the infection.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprised of FIGS. 1A-1G, depicts how systemic administration oftyphoid toxin causes symptoms observed during the acute phase of typhoidfever. FIG. 1A depicts an exemplary chromatographic profile of thetyphoid toxin holotoxin used in the biological assays. The inset shows aCoomassie blue stained SDS-PAGE analysis of the peak fraction shown onthe chromatogram. FIG. 1B depicts how typhoid toxin induces cell cyclearrest in cultured cells. Human intestinal epithelial Henle-407 cellswere left untreated, or treated for 48 hrs with 0.02 nM of the purifiedtyphoid toxin. The cell cycle profiles were then analyzed by flowcytometry. The insets show representative light microscope images ofmock or typhoid toxin treated Henle-407 cells 48 hrs after treatment.FIG. 1C depicts how typhoid toxin induces cell cycle arrest in culturedcells. Averages of cell cycle profiles from at least 3 independentexperiments. Bar represents average ± standard deviation. ***, P<0.001,compared to the number of cells in G2M of the control untreated group.UT: untreated; TT: typhoid toxin treated. FIG. 1D depicts weight lossafter treatment with different typhoid toxin preparations. The indicatedtyphoid toxin preparations were administered intravenously to groups ofC57BL/6 and 5 days after treatment animals were weighed. Lines are themean ± standard error of the mean and represent the weight relative tothe values before treatment. ***, P<0.0001. FIG. 1E depicts the survivalof animals receiving different typhoid toxin preparations. n=3-5 animalsper group. FIG. 1F depicts how typhoid toxin causes neutrophildepletion. Circulating white blood cells were counted in a hematologyanalyzer (*, P<0.05). FIG. 1G depicts how typhoid toxin causesneutrophil depletion. Alternatively, peripheral blood cells from animalsthat have received the indicated treatment were stained with an antibodydirected to the neutrophil cell marker Grl and the number of stainedcells was determined by flow cytometry. Numbers of neutrophils (verticaldashed line) were significantly reduced by typhoid toxin injection. Thehistograms shown are from ungated samples. Similar results were obtainedin several independent repetitions of the experiment. RFI, relativefluorescence intensity.

FIG. 2, comprised of FIGS. 2A-2H, depicts how typhoid toxin recognizesterminally sialylated glycans on surface glycoproteins of target cells.FIG. 2A depicts affinity purification of typhoid toxin-interactingsurface proteins. Henle-407 cell surface proteins were biotinylated,co-immunoprecipitated with purified typhoid toxin (TT), and analyzed bySDS-PAGE. FIG. 2B depicts affinity purification of typhoidtoxin-interacting surface proteins. Henle-407 cell surface proteins werebiotinylated, co-immunoprecipitated with purified typhoid toxin (TT),and analyzed by LC-MS/MS. The peptides from Podocalixin like protein 1(PODXL) (indicated by an asterix in FIG. 2A) identified by LC-MS/MS areindicated in bold, the shaded sequence indicates the position of itstransmembrane domain, and the underlined sequence its signal peptide.Note that a majority of identified peptides are from the C-terminalregion because the heavy glycosylation of the N-terminus extracellularregion of Podocalyxin interferes with the LC-MS/MS analysis. FIG. 2Cdepicts how PODXL depletion reduces toxin binding and toxicity.PODXL-depleted (by an specifically targeted siRNA) and control cellswere treated with fluorescently-labeled typhoid toxin and toxin bindingwas evaluated by flow cytometry (* P=0.024 for three independentdeterminations). FIG. 2D depicts how PODXL depletion reduces toxinbinding and toxicity. siRNA-depleted and control cells were treated withtyphoid toxin and subjected to flow cytometric cell cycle analysis (toevaluate toxicity). FIG. 2E depicts how the removal of surface glycansreduces toxin binding. Henle-407 cells were treated with a mixture ofglycosidases and the ability of treated and control cells to bindfluorescently-labeled toxin was subsequently evaluated by flow cytometry(***, P<0.001 from three independent experiments). FIG. 2F depicts how amutant cell line lacking surface N-glycans is resistant to typhoidtoxin. The N-acetylglucosaminyltransferase I-deficient (Lec1) and itsparent (Pros) cell lines were treated with typhoid toxin and toxicitywas evaluated by flow cytometric cell cycle analysis. FIG. 2G depictsthe averages of cell cycle profiles from several independent experimentsexemplified in FIG. 2F. Bar represents average ± standard deviation ofat least three independent determinations. **, P<0.01, compared to thenumber of Pro5 cells in G2M. FIG. 2H depicts an exemplary glycan arrayanalysis of typhoid toxin binding. Included in this array were 610glycans and the highest and lowest points from each set of 6 replicateshave been removed so the average RFU (relative fluorescence unit) is of4 values. The X axis depicts the glycan numbers. The structure of themost relevant glycans is shown. The raw data are shown in FIGS. 20 and21.

FIG. 3, comprised of FIGS. 3A-3E, depicts how the crystal structure oftyphoid toxin depicts a unique architecture. FIG. 3A depicts two viewsof the overall structure of the typhoid holotoxin complex shown as aribbon cartoon and related by 90° rotation about a vertical axis. CdtB,PltA and PltB are shown. FIG. 3B depicts a bottom view of the channelformed by the PltB pentamer, depicting the PltA C-terminal a-helixwithin it. FIG. 3C depicts the surface charge distribution of thepredicted sugar-binding pockets of different B subunit homologs of theindicated AB₅ toxins (SubB for Subtilase and S2 for Pertussis toxins). Ahighly conserved serine residue critical for sugar binding is indicatedwithin the sugar-binding pocket. The sugars N-glycolylneuraminic acid(within SubB) and N-acetylneuraminic acid (within typhoid and pertussistoxins) are shown. FIG. 3D depicts molecular modeling ofN-acetylneuraminic acid within the typhoid and pertussis toxins bindingpocket. Critical residues engaged in this interaction are shown. FIG. 3Edepicts the atomic interface between CdtB and PltA. The inset shows adetailed view of a critical disulfide bond between PltA Cys214 and CdtBCys269.

FIG. 4, comprised of FIGS. 4A-4I, depicts an exemplarystructure-function analysis of typhoid toxin. FIGS. 4A-4D depicts howSer35 on the predicted PltB glycan-binding site is critical for typhoidtoxin binding and toxicity. FIG. 4A depicts how fluorescently labeledtyphoid toxin containing PltBSer35A was tested for its binding toglycans. FIG. 4B depicts how fluorescently labeled typhoid toxincontaining PltBSer35A was tested for its binding to cultured epithelialcells. (For FIGS. 4A and 4B, see FIG. 2 legend for details; raw data togenerate panel A is shown in Table 1) (***, P<0.001 from at least threeindependent determinations). FIG. 4C depicts how the toxicity offluorescently labeled typhoid toxin was assayed by flow cytometric cellcycle analysis of toxin-treated cultured epithelial cells. FIG. 4Ddepicts how the toxicity of fluorescently labeled typhoid toxin wasassayed by systemic administration to C57BL/6 mice (n=3 to 5 mice).Equivalent results were observed in several independent experiments.FIGS. 4E-4I depict how the disulfide bond between PltA and CdtB isessential for typhoid toxin complex formation in vitro and toxicity invivo. FIG. 4E depicts how the typhoid toxin complex was analyzed by ionexchange chromatography before and after treatment with DTT (L: loadingcontrol; M: molecular weight markers; F: chromatographic fraction). Thedifferent fractions were then analyzed by SDS-PAGE (shown in the inset)for the presence of the different components of typhoid toxin (i.e.,PltB, CdtB, and PltA). Treatment with DTT resulted in the release ofCdtB from the PltA/PltB complex. FIG. 4F depicts how CdtB ACys269 is notincorporated into the typhoid toxin complex. A typhoid toxin preparationobtained from a bacterial strain expressing CdtB ACys269 was analyzed bygel filtration chromatography and compared to wild-type toxin. Whilewild-type holotoxin eluted in fractions 13 and 14, toxin obtained from abacterial strain encoding CdtB ΔCys269 eluted in fractions 14 and 15 dueto the loss of its CdtB subunit. FIG. 4G depicts how Henle-407 cellsinfected with S. typhi strains were examined for toxicity by flowcytometric cell cycle analysis. FIG. 4H depicts how Henle-407 cellsinfected with S. typhi strains were fixed 24 hours after infectioncells, stained with anti-FLAG antibody, and imaged in a fluorescencemicroscope. The puncta staining, which represent CdtB in typhoid toxinexport carriers, are not observed in cells infected with the strain thatexpresses CdtB ACys269. FIG. 4I depicts the quantification of punctastaining The values shown represent averages of puncta intensities ininfected cells. Bar represents average ± standard error means. At least100 cells were examined. Scale bar:10 μm. FIG. 4J depicts how thecritical cysteines that tether CdtB to PltA are absent in closehomologs. ClustalW amino acid sequence comparison analyses of CdtB andPltA homologs. The CdtB homologs (and Genbank entry numbers) used in thealignment were from Shigella boydii (AAU88264.1), Providenciaalcalifaciens (BAL72684.1), Helicobacter hepaticus (AAF19158.1),Haemophilus ducreyi (NP 873398.1), E. coli (BAH72965.1), Campylobacterjejuni (AAS01598.1), and Aggregatibacter actinomycetemcomitans(AAC70898.1). The PltA homolog used in the alignment was the highlyrelated S. typhimurium DT104 ArtA (BAE20153.1). Conserved and uniquecysteines are shown.

FIG. 5 is a graph depicting body temperature of typhoid toxin or buffertreated animals. Each dot represents a measurement of a single animal.

FIG. 6 is a series of graphs depicting how typhoid toxin is able tointoxicate a broad range of host cells. Various host cells were mocktreated or treated with 0.02 nM of purified typhoid holotoxin for 24hours (i.e., Raw, Jurkat, and Ramos cells) or for 48 hours (i.e., COS1,CHO, MDCK, and NIH3T3) and the cell cycle profiles of the treated cellsdetermined by flow cytometric analysis. Equivalent results were obtainedin several repetitions of this experiment.

FIG. 7 is a graph depicting podxl mRNA levels in control cells or cellexpressing an siRNA targeted to podxl as measured by real-time PCR. Barrepresents the average of the expression level ± standard deviation ofthree independent determinations.

FIG. 8, comprising FIGS. 8A-8B, depicts how typhoid toxin recognizesterminally sialylated glycans on CD45 in T, B, and macrophage celllines. Cell surface proteins from Jurkat, Ramos, and THP1 cells werebiotinylated, co-immunoprecipitated with purified typhoid toxin (TT),and analyzed by SDS-PAGE and LC-MS/MS. FIG. 8A is a photograph of a geldepicting Jurkat cells analyzed by SDS-PAGE. FIG. 8B is an imagedepicting the CD45 peptides identified by LCMS/MS. The CD45 peptides areindicated as underlined for Jurkat, shaded boxes for Ramos, and THP1cells. The location of the signal peptide is indicated in italics.

FIG. 9 is a series of images depicting how a mutant cell line that lackssurface N-glycans is more resistant to typhoid toxin. TheN-acetylglucosaminyltransferase I-deficient (Lec1) and its parent (Pro5)cell lines were treated with typhoid toxin and examined by lightmicroscopy. Cell distention is observed in Pro5, but not in Lecl toxintreated cells. Scale bar: 50 μm.

FIG. 10 is a graph depicting how a mutant cell line that lacks surfaceN-glycans can be intoxicated by typhoid toxin when administered at highconcentrations. The N-acetylglucosaminyltransferase I-deficient (Lec1)and its parent (Pro5) cell lines were treated with increasingconcentrations of typhoid toxin (as indicated) and toxicity wasevaluated by flow cytometric cell cycle analysis 36 hours aftertreatment. Bar presents average ± standard deviation of at least threeindependent determinations. *, P<0.05, **P<0.01 compared to the numberof cells in G2M of untreated (UT) group.

FIG. 11, comprised of FIGS. 11A-11C, depicts how typhoid toxin exhibitsan A₂B₅ composition. FIG. 11A is a table depicting all the possiblecomplexes compatible with the observed molecular weight of typhoid toxin(116 kDA as measured by SEC-LS analysis). FIG. 11B is a table depictinghow complex 1 is the most likely for the observed extinctioncoefficient. FIG. 11C is a table depicting amino acid compositionanalysis of typhoid toxin. The purified typhoid holotoxin complex wasresolved on a 15% SDS-PAGE gel, stained with coomassie brilliant blue,and the three individual bands were excised for quantitative amino acidanalysis.

FIG. 12 is an illustration depicting structure alignment between typhoidtoxin PltA and pertussis toxin 51. The conserved catalytic residueglutamic acid and disulfide bond are shown in inset.

FIG. 13 is an illustration depicting structure alignment between CdtBfrom typhoid toxin and from H. ducreyi CDT. The conserved catalytic(His160 and His 274) and DNA-contact (Arg117, Arg144, and Asn201) areshown.

FIG. 14 is an illustration depicting structure alignment of PltBhomologs from the B subunits of Subtilase (SubB) and Pertussis (S2)toxins. A conserved serine essential for sugar binding is depicted inthe inset showing the different structural elements surrounding thiscritical residue (a loop in PltB and S2, and a β-strand in SubB).

FIG. 15 is a series of images depicting conserved sugar-binding residuesin typhoid toxin PltB and pertussis toxin S2. The position of the sugarligand N-acetylneuraminic acid (Neu5Ac) relative to key residues ofpertussis toxin S2 (from the crystal structure) and PltB (from moleculardocking) is depicted.

FIG. 16 is a series of graphs depicting how Ser35 is critical fortyphoid toxin glycan binding. Surface plasmon resonance assay of thebinding of wild type (WT) and PltBS35A typhoid toxin preparations to theGD2 glycan. Numbers on the y axis depict the relative response units(RU). Binding curves were generated by averaging the values severalindependent determinations. The observed Rmax suggests that ˜50% ofprotein remains active when captured by an anti-flag antibody. Althoughnot wishing to be bound by any particular theory, such Rmax valuesstrongly suggest that, for wild type, on average there are 2.5 sugarmolecules per PltB pentamer, assuming that each monomer is 100% activefor binding.

FIG. 17 is a series of illustrations depicting surface chargedistribution of the PltB pentamer depicting its hydrophobic channel(left panel). The interaction of key PltA residue with the lumen of thePltB channel is shown in detail (right panel).

FIG. 18 is a graph depicting how an S. typhi CdtB ΔCys269 mutant doesnot intoxicate culture cells. Henle-407 cells were left uninfected orinfected for 4 days with wild type S. typhi or a CdtB ΔCys269 mutant atvarious multiplicity of infections (moi) as indicated. Typhoid toxinmediated toxicity was evaluated by flow cytometric cell cycle analysis.Bar represents the average ± standard deviation. ***, P<0.001 comparedto the number of cells in G2M in the uninfected (UI) group.

FIG. 19 is an image of a gel depicting expression of CdtB ACys269 in S.typhi infected culture epithelial cells. Henle-407 cells were infectedwith S. typhi strains expressing FLAG-epitope tag wild type CdtB or CdtBACys269 and 24 hrs after infection, the levels of CdtB in cell lysateswas investigated by western blot analysis. The bacterial protein DnaKwas used as a loading control.

FIG. 20, comprised of FIGS. 20A-20C, is a series of tables depictingglycans showing typhoid toxin binding activity classified by theirstructural features.

FIG. 21, comprised of FIGS. 21A-21R, is a series of tables depictingglycan array analysis for binding to typhoid toxin. The highlighted areain grey depicts glycans present in glycolipids. Average RFU indicatesthe average fluorescent unites from 4 independent determinations. STDv:standard deviation; % CV: variation coefficient.

FIG. 22, comprised of FIGS. 22A-22R, is a series of tables depictingglycan array analysis for binding to the typhoid toxin Ser53A mutant.Average RFU indicates the average fluorescent unites from 4 independentdeterminations. STDv: standard deviation; % CV: variation coefficient.

FIG. 23 depicts the results of experiments demonstrating that Typhoidtoxin stimulates the production of neutralizing antibodies in mice. Mice(4 animals per group) were immunized with a typhoid toxin toxoidpreparation, or a typhoid toxin preparation containing a glycan-bindingdeficient B subunit (PltBS35A), alone or in combination with an adjuvant(Alumn). Sera were collected from immunized animals 4 weeks afterimmunization and tested for levels of antibody by ELISA (left panel). Inaddition, the sera of immunized animals were tested for the presence oftyphoid toxin neutralizing antibodies (left panel) as previouslydescribed (Spano et al., 2009, Cell Host Microbe 3:30-38). Briefly,cultured cells were infected with S. Typhi, left untreated (control) ortreated with antibodies obtained from non-immunized animals (−), animalsimmunized with the indicated preparations. Forty-eight hours afterinfection, the DNA content of cells (to determine cell cycle stage) wasdetermined by flow cytometry.

FIG. 24 depicts the results of experiments assessing anti-typhoid toxinserum antibody levels in a convalescent and a control population.

DETAILED DESCRIPTION

The present invention provides compositions and methods to prevent,treat and diagnose infection by Salmonella enterica serovar typhi (S.typhi) and/or S. paratyphi, i.e., typhoid fever. In one embodiment, thecomposition of the invention is a vaccine that induces the cell-mediatedand/or humoral immunity directed against at least one S. typhi and/or S.paratyphi protein (e.g., PltA, PltB, CdtB, etc). In one embodiment, thecomposition comprises PltA, or a mutant thereof. In one embodiment, thecomposition comprises PltB, or a mutant thereof. In one embodiment, thecomposition comprises CdtB, or a mutant thereof.

In another embodiment, the composition of the invention comprises anucleic acid sequence encoding PltA, or a mutant thereof. In anotherembodiment, the composition of the invention comprises a nucleic acidsequence encoding PltB, or a mutant thereof. In one embodiment, thecomposition of the invention comprises a nucleic acid sequence encodingCdtB, or a mutant thereof.

In one embodiment, the composition comprises an antibody thatspecifically binds to PltA, or a mutant thereof. In one embodiment, thecomposition comprises an antibody that specifically binds to PltB, or amutant thereof. In one embodiment, the composition comprises an antibodythat specifically binds to CdtB, or a mutant thereof.

The invention provides methods of inducing an immune response forpreventing or treating infection by S. typhi or S. paratyphi. In oneembodiment, the methods comprise administering at least one of PltA,PltB, CdtB, or mutants thereof, to a subject. In another embodiment, themethods comprise administering a nucleic acid encoding at least one ofPltA, PltB, CdtB, or mutants thereof, to a subject.

In another embodiment, the methods comprise administering a nucleic acidencoding and expressing at least one of PltA, PltB, CdtB, or mutantsthereof, to a bacterium. In some embodiments, the bacterium alreadycomprises a nucleic that encodes, but does not express, PltA, PltB,CdtB, or mutants thereof. In other embodiments, the bacterium alreadycomprises a nucleic that encodes, and does express, PltA, PltB, CdtB, ormutants thereof.

The invention also provides methods of treating infection by S. typhi orS. paratyphi in a subject in need thereof. In one embodiment, the methodcomprises administering to the subject at least one antibody thatspecifically binds to PltA, or a mutant thereof. In one embodiment, themethod comprises administering to the subject at least one antibody thatspecifically binds to PltB, or a mutant thereof. In one embodiment, themethod comprises administering to the subject at least one antibody thatspecifically binds to CdtB, or a mutant thereof. In one embodiment, themethod comprises administering to the subject at least two antibodiesthat specifically bind to at least two of PltA, PltB and CdtB, ormutants thereof.

The invention also includes inhibitor compositions and methods forinhibiting with the interaction between the S. typhi or S. paratyphitoxin and the toxin's receptor.

The invention also provides methods of diagnosing infection by S. typhior S. paratyphi in a subject by detecting the presence of, or measuringthe level of, in the subject, at least one of PltA, PltB, CdtB, orantibodies that specifically bind to at least one of PltA, PltB, CdtB,or mutants thereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.The antibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York;Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird etal., 1988, Science 242:423-426).

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample.

As used herein, the term “autologous” is meant to refer to any materialderived from an individual to which it is later to be re-introduced intothe same individual.

The term “agent” includes any substance, metabolite, molecule, element,compound, or a combination thereof. It includes, but is not limited to,e.g., protein, oligopeptide, small organic molecule, glycan,polysaccharide, polynucleotide, and the like. It can be a naturalproduct, a synthetic compound, a chemical compound, or a combination oftwo or more substances. Unless otherwise specified, the terms “agent,”“substance,” and “compound” can be used interchangeably. Further, a“test agent” or “candidate agent” is generally a subject agent for usein an assay of the invention.

The term “binding” refers to a direct association between at least twomolecules, due to, for example, covalent, electrostatic, hydrophobic,ionic and/or hydrogen-bond interactions.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the antigen binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature342, p 877-883.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

“Contacting” refers to a process in which two or more molecules or twoor more components of the same molecule or different molecules arebrought into physical proximity such that they are able undergo aninteraction. Molecules or components thereof may be contacted bycombining two or more different components containing molecules, forexample by mixing two or more solution components, preparing a solutioncomprising two or more molecules such as target, candidate orcompetitive binding reference molecules, and/or combining two or moreflowing components.

As used herein, by “combination therapy” is meant that a first agent isadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual. Suchcombinations are considered to be part of a single treatment regimen orregime.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap temporally with each other.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “donor antibody” refers to an antibody (monoclonal, and/orrecombinant) which contributes the amino acid sequences of its variableregions, CDRs, or other functional fragments or analogs thereof to afirst immunoglobulin partner, so as to provide the alteredimmunoglobulin coding region and resulting expressed altered antibodywith the antigenic specificity and neutralizing activity characteristicof the donor antibody.

The term “acceptor antibody” refers to an antibody (monoclonal and/orrecombinant) heterologous to the donor antibody, which contributes all(or any portion, but in some embodiments all) of the amino acidsequences encoding its heavy and/or light chain framework regions and/orits heavy and/or light chain constant regions to the firstimmunoglobulin partner. In certain embodiments a human antibody is theacceptor antibody.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

As used herein, the term “heavy chain antibody” or “heavy chainantibodies” comprises immunoglobulin molecules derived from camelidspecies, either by immunization with a peptide and subsequent isolationof sera, or by the cloning and expression of nucleic acid sequencesencoding such antibodies. The term “heavy chain antibody” or “heavychain antibodies” further encompasses immunoglobulin molecules isolatedfrom an animal with heavy chain disease, or prepared by the cloning andexpression of VH (variable heavy chain immunoglobulin) genes from ananimal. “Homologous” refers to the sequence similarity or sequenceidentity between two polypeptides or between two nucleic acid molecules.When a position in both of the two compared sequences is occupied by thesame base or amino acid monomer subunit, e.g., if a position in each oftwo DNA molecules is occupied by adenine, then the molecules arehomologous at that position. The percent of homology between twosequences is a function of the number of matching or homologouspositions shared by the two sequences divided by the number of positionscompared multiplied by 100. For example, if 6 of 10 of the positions intwo sequences are matched or homologous then the two sequences are 60%homologous. By way of example, the DNA sequences ATTGCC and TATGGC share50% homology. Generally, a comparison is made when two sequences arealigned to give maximum homology.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., 1989, Queen etal., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al.,Bio/Technology, 9:421). A suitable human acceptor antibody may be oneselected from a conventional database, e.g., the KABAT database, LosAlamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanized antibodies (see for example EP-A-0239400 andEP-A-054951).

The term “immunoglobulin” or “Ig,” as used herein, is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, the term “immune response” includes T-cell mediatedand/or B-cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity, and B cell responses, e.g., antibody production. Inaddition, the term immune response includes immune responses that areindirectly affected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages. Immune cells involved in the immune response includelymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2cells); antigen presenting cells (e.g., professional antigen presentingcells such as dendritic cells, macrophages, B lymphocytes, Langerhanscells, and non-professional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmacrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, an “inhibitory-effective amount” is an amount thatresults in a detectable (e.g., measurable) amount of inhibition of anactivity. In some instance, the activity is its ability to bind withanother component.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

A “mutation,” as used herein, refers to a change in nucleic acid orpolypeptide sequence relative to a reference sequence (which ispreferably a naturally-occurring normal or “wild-type” sequence), andincludes translocations, deletions, insertions, and substitutions/pointmutations. A “mutant” as used herein, refers to either a nucleic acid orprotein comprising a mutation.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintradermal (i.d.) injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “X,”the presence of a molecule containing epitope X (or free, unlabeled A),in a reaction containing labeled “X” and the antibody, will reduce theamount of labeled X bound to the antibody.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression,diminution, remission, or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or clinical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

A term “toxoid” as used herein, refers to a bacterial toxin, thetoxicity of which has been inactivated or suppressed, such as byintroduction of a mutation, a chemical treatment, or a heat treatment,while other properties of the toxin, such as immunogenicity, aremaintained in the toxoid. In some literature, toxoids are referred to asanatoxins.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The invention relates to the administration of at least one of PltA,PltB, CdtB, or mutants thereof, of S. typhi or and S. paratyphi to asubject to induce an immune response. Thus, the present inventionprovides a polypeptide or a combination of polypeptides, apolynucleotide or a combination of polynucleotides, which are useful ininducing an immune response, for the treatment or prevention ofinfection by S. typhi or S. paratyphi.

The invention provides an immunological composition comprising apolypeptide or combination of polypeptides derived from at least one ofPltA, PltB, CdtB, or mutants thereof, useful in eliciting an immuneresponse. The compositions comprising one or more polypeptides of theinvention not only are useful as a prophylactic therapeutic agent forimmunoprotection, but are also useful as a therapeutic agent fortreatment of an ongoing condition associated with infection by S. typhior S. paratyphi in a subject.

In one embodiment, the composition of the invention comprises a nucleicacid sequence encoding PltA, or a mutant thereof. In one embodiment, thePltA mutant is PltA E133X. In another embodiment, the PltA mutant isPltA E133A.

In another embodiment, the composition of the invention comprises anucleic acid sequence encoding PltB, or a mutant thereof. In oneembodiment, the PltB mutant is PltB S35X. In another embodiment, thePltB mutant is PltB S35A.

In one embodiment, the composition of the invention comprises a nucleicacid sequence encoding CdtB, or a mutant thereof In one embodiment, theCdtB mutant is CdtB H160X. In another embodiment, the CdtB mutant isCdtB H160Q. In one embodiment, the CdtB mutant is CdtB R119X. In oneembodiment, the CdtB mutant is CdtB H259X. In one embodiment, the CdtBmutant is CdtB ΔCys269. In another embodiment, the CdtB mutant is CdtBC269X.

In one embodiment, the mutant PltA comprises any mutation in PltA thatdisrupts its enzymatic activity. In one embodiment, the mutant PltBcomprises any mutation in PltB that disrupts its enzymatic activity. Inone embodiment, the mutant CdtB comprises any mutation in CdtB thatdisrupts its enzymatic activity.

The skilled artisan will understand that the least one of PltA, PltB,CdtB, or mutants thereof, useful in eliciting an immune response, caneach be used alone or in any combination for eliciting an immuneresponse.

The present invention also provides methods of preventing, inhibiting,and treating infection by S. typhi or S. paratyphi in a subject in needthereof In one embodiment, the methods of the invention induce immunityagainst S. typhi or S. paratyphi in the subject, by generating an immuneresponse in the subject directed to at least one polypeptide, such asPltA, PltB and CdtB. In one embodiment, the methods of the inventioninduce production of PltA-specific antibodies in the subject. In oneembodiment, the methods of the invention induce production ofPltB-specific antibodies in the subject. In one embodiment, the methodsof the invention induce production of CdtB-specific antibodies in thesubject.

In one embodiment, the methods of the invention prevent S. typhi or S.paratyphi related pathology in a subject in need thereof. In oneembodiment, the methods of the invention comprise administering to thesubject a composition comprising at least a portion of at least one ofPltA, PltB, CdtB, or mutants thereof, to a subject. In anotherembodiment, the methods of the invention comprise administering to thesubject a composition comprising a nucleic acid sequence encoding atleast one of PltA, PltB, CdtB, or mutants thereof, to a subject. Invarious embodiments, the composition can be comprise a single subunit ofA₂B₅, a combination of subunits of A₂B₅, or the entire A₂B₅, wherein atleast one of the subunits is a mutant subunit.

In another embodiment, the methods of the invention compriseadministering to the subject a bacterium or virus comprising a nucleicacid sequence encoding at least one of PltA, PltB, CdtB, or mutantsthereof. In another embodiment, the methods of the invention compriseadministering to the subject a bacterium or virus expressing at least aportion of at least one of PltA, PltB, CdtB, or mutants thereof. Inanother embodiment, the methods of the invention comprise administeringto the subject a bacterium or virus comprising at least a portion of atleast one of PltA, PltB, CdtB, or mutants thereof.

The invention also includes inhibitor compositions and methods forinhibiting with the interaction between the S. typhi or S. paratyphitoxin and the toxin's receptor.

The invention also provides methods of diagnosing infection by S. typhior S. paratyphi in a subject by detecting the presence of, or measuringthe level of, in the subject, at least one of PltA, PltB, CdtB, ormutants thereof, or antibodies that specifically bind to at least one ofPltA, PltB, CdtB, or mutants thereof.

Compositions

The present invention provides compositions, including polypeptides,nucleotides, vectors, and vaccines, that when administered to a subject,elicit an immune response directed against S. typhi or S. paratyphi,including an immune response directed against at least one of PltA,PltB, CdtB, or mutants thereof. Further, when the compositions areadministered to a subject, they elicit an immune response that serves toprotect the inoculated subject against conditions associated with S.typhi or S. paratyphi infection.

In one embodiment, the present invention provides compositions that areuseful as immunomodulatory agents, for example, in stimulating immuneresponses and in preventing S. typhi or S. paratyphi related pathology.In various embodiments, the immunomodulatory agents comprise at leastone of PltA, PltB, CdtB, or mutants thereof. In one embodiment, theimmune response is not detrimental to the host and therefore thecompositions of the invention are useful as a vaccine. In oneembodiment, the immunomodulatory agents are administered in combinationwith an adjuvant. In another embodiment, the immunomodulatory agents areadministered in the absence of an adjuvant.

PltA, PltB, CdtB, or mutants thereof can be, used as immunostimulatoryagents to induce the production of specific antibodies and protectagainst S. typhi or S. paratyphi induced pathology. Therefore, in oneembodiment, the composition of the invention comprises a PltApolypeptide, or a mutant thereof. In one embodiment, the PltA mutant isPltA E133X. In another embodiment, the PltA mutant is PltA E133A. Inanother embodiment, the composition of the invention comprises a PltBpolypeptide, or a mutant thereof. In one embodiment, the PltB mutant isPltB S35X. In another embodiment, the PltB mutant is PltB S35A. Inanother embodiment, the composition of the invention comprises a CdtBpolypeptide, or a mutant thereof. In one embodiment, the CdtB mutant isCdtB H160X. In another embodiment, the CdtB mutant is CdtB H160Q. In oneembodiment, the CdtB mutant is CdtB R119X. In another embodiment, theCdtB mutant is CdtB H259X. In one embodiment, the CdtB mutant is CdtBACys269. In another embodiment, the CdtB mutant is CdtB C269X. Theskilled artisan will understand that the least one of PltA, PltB, CdtB,or mutants thereof, useful in eliciting an immune response, can each beused alone or in any combination for eliciting an immune response.

The present invention also provides polynucleotides that encode thepolypeptides described herein. Therefore, in one embodiment, thecomposition of the invention comprises a nucleic acid sequence encodingPltA, or a mutant thereof. In one embodiment, the PltA mutant is PltAE133X. In another embodiment, the PltA mutant is PltA E133A. In anotherembodiment, the composition of the invention comprises a nucleic acidsequence encoding PltB, or a mutant thereof. In one embodiment, the PltBmutant is PltB S35X. In another embodiment, the PltB mutant is PltBS35A. In one embodiment, the composition of the invention comprises anucleic acid sequence encoding CdtB, or a mutant thereof. In oneembodiment, the CdtB mutant is CdtB H160X. In another embodiment, theCdtB mutant is CdtB H160Q. In one embodiment, the CdtB mutant is CdtBR119X. In another embodiment, the CdtB mutant is CdtB H259X. In oneembodiment, the CdtB mutant is CdtB ACys269. In another embodiment, theCdtB mutant is CdtB C269X. The skilled artisan will understand that theleast one of PltA, PltB, CdtB, or mutants thereof, useful in elicitingan immune response, can each be used alone or in any combination foreliciting an immune response.

In various embodiments, the invention provides a polypeptide, or afragment of a polypeptide, a homolog, a mutant, a variant, a derivativeor a salt of a polypeptide as elsewhere described herein, wherein theimmunogenic activity of the polypeptide or fragment thereof is retained.

The invention should also be construed to include any form of apolypeptide having substantial homology to the polypeptides disclosedherein. Preferably, a polypeptide which is “substantially homologous” isabout 50% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous, more preferably about 90% homologous,even more preferably, about 95% homologous, and even more preferablyabout 99% homologous to amino acid sequence of the polypeptidesdisclosed herein.

In one embodiment, the polypeptide or combination of polypeptides of thepresent invention are capable of generating a specific immune response.In another embodiment, the polypeptide or combination of polypeptides ofthe present invention are capable of generating specific antibodies.

Polypeptides of the present invention can be prepared using well knowntechniques. For example, the polypeptides can be prepared synthetically,using either recombinant DNA technology or chemical synthesis.Polypeptides of the present invention may be synthesized individually oras longer polypeptides composed of two or more polypeptides. Thepolypeptides of the present invention can be isolated, i.e.,substantially free of other naturally occurring host cell proteins andfragments thereof.

The polypeptides of the present invention may contain modifications,such as glycosylation, aglycosylation, side chain oxidation, orphosphorylation; so long as the modifications do not destroy theimmunologic activity of the polypeptides. Other modifications includeincorporation of D-amino acids or other amino acid mimetics that can beused, for example, to increase the serum half-life of the polypeptides.

The polypeptides of the invention can be modified whereby the amino acidis substituted for a different amino acid in which the properties of theamino acid side-chain are conserved (a process known as conservativeamino acid substitution).

Examples of properties of amino acid side chains are hydrophobic aminoacids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C,E, Q, G, H, K, S, T), and side chains having the following functionalgroups or characteristics in common: an aliphatic side-chain (G, A, V,L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfuratom containing side-chain (C, M); a carboxylic acid and amidecontaining side-chain (D,

N, E, Q); a base containing side-chain (R, K, H); and an aromaticcontaining side-chain (H, F, Y, W). Note that the parenthetic lettersindicate the one-letter codes of amino acids. As used herein, X standsfor any amino acid.

The polypeptides of the invention can be prepared as a combination,which includes two or more of polypeptides of the invention, for use asa vaccine for prevention or treatment of S. typhi or S. paratyphiinfection. The polypeptides may be in a cocktail or may be conjugated toeach other using standard techniques. For example, the polypeptides canbe expressed as a single polypeptide sequence. The polypeptides in thecombination may be the same or different.

The present invention should also be construed to encompass “mutants,”“derivatives,” and “variants” of the polypeptides of the invention (orof the DNA encoding the same) which mutants, derivatives and variantsare polypeptides which are altered in one or more amino acids (or, whenreferring to the nucleotide sequence encoding the same, are altered inone or more base pairs) such that the resulting polypeptide (or DNA) isnot identical to the sequences recited herein, but has the samebiological property as the polypeptides disclosed herein.

The nucleic acid sequences include both the DNA sequence that istranscribed into RNA and the RNA sequence that is translated into apolypeptide. According to other embodiments, the polynucleotides of theinvention are inferred from the amino acid sequence of the polypeptidesof the invention. As is known in the art several alternativepolynucleotides are possible due to redundant codons, while retainingthe biological activity of the translated polypeptides.

Further, the invention encompasses an isolated nucleic acid encoding apolypeptide having substantial homology to the polypeptides disclosedherein. Preferably, the nucleotide sequence of an isolated nucleic acidencoding a polypeptide of the invention is “substantially homologous,”that is, is about 60% homologous, more preferably about 70% homologous,even more preferably about 80% homologous, more preferably about 90%homologous, even more preferably, about 95% homologous, and even morepreferably about 99% homologous to a nucleotide sequence of an isolatednucleic acid encoding a polypeptide of the invention.

It is to be understood explicitly that the scope of the presentinvention encompasses homologs, analogs, variants, fragments,derivatives and salts, including shorter and longer polypeptides andpolynucleotides, as well as polypeptide and polynucleotide analogs withone or more amino acid or nucleic acid substitution, as well as aminoacid or nucleic acid derivatives, non-natural amino or nucleic acids andsynthetic amino or nucleic acids as are known in the art, with thestipulation that these modifications must preserve the immunologicactivity of the original molecule. Specifically any active fragments ofthe active polypeptides as well as extensions, conjugates and mixturesare included and are disclosed herein according to the principles of thepresent invention.

The invention should be construed to include any and all isolatednucleic acids which are homologous to the nucleic acids described andreferenced herein, provided these homologous nucleic acids encodepolypeptides having the biological activity of the polypeptidesdisclosed herein.

The skilled artisan would understand that the nucleic acids of theinvention encompass an RNA or a DNA sequence encoding a polypeptide ofthe invention, and any modified forms thereof, including chemicalmodifications of the DNA or RNA which render the nucleotide sequencemore stable when it is cell free or when it is associated with a cell.Chemical modifications of nucleotides may also be used to enhance theefficiency with which a nucleotide sequence is taken up by a cell or theefficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

Further, any number of procedures may be used for the generation ofmutant, derivative or variant forms of a protein of the invention usingrecombinant DNA methodology well known in the art such as, for example,that described in Sambrook et al. (2012, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al.(1997, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork). Procedures for the introduction of amino acid changes in apolypeptide or polypeptide by altering the DNA sequence encoding thepolypeptide are well known in the art and are also described in these,and other, treatises.

Vectors

The nucleic acids encoding the polypeptide or combinations ofpolypeptides of the invention of the invention can be incorporated intosuitable vectors, including but not limited to, plasmids and retroviralvectors. Such vectors are well known in the art and are therefore notdescribed in detail herein. In one embodiment, the invention includes anucleic acid sequence encoding one or more polypeptides of the inventionoperably linked to a nucleic acid comprising a promoter/regulatorysequence such that the nucleic acid is preferably capable of directingexpression of the protein encoded by the nucleic acid. Thus, theinvention encompasses expression vectors and methods for theintroduction of exogenous DNA into cells with concomitant expression ofthe exogenous DNA in the cells such as those described, for example, inSambrook et al. (2012, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York).The incorporation of a desired polynucleotide into a vector and thechoice of vectors is well-known in the art as described in, for example,Sambrook et al. (2012), and in Ausubel et al. (1997).

The polynucleotide can be cloned into a number of types of vectors.However, the present invention should not be construed to be limited toany particular vector. Instead, the present invention should beconstrued to encompass a wide plethora of vectors which are readilyavailable and/or well-known in the art. For example, the polynucleotideof the invention can be cloned into a vector including, but not limitedto a plasmid, a phagemid, a phage derivative, an animal virus, and acosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

In specific embodiments, the expression vector is selected from thegroup consisting of a viral vector, a bacterial vector and a mammaliancell vector. Numerous expression vector systems exist that comprise atleast a part or all of the compositions discussed above. Prokaryote-and/or eukaryote-vector based systems can be employed for use with thepresent invention to produce polynucleotides, or their cognatepolypeptides. Many such systems are commercially and widely available.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2012), and in Ausubel et al.(1997), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193.

For expression of the desired nucleotide sequences of the invention, atleast one module in each promoter functions to position the start sitefor RNA synthesis. The best known example of this is the TATA box, butin some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 genes, a discrete element overlying the start site itselfhelps to fix the place of initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 by upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50by apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either co-operativelyor independently to activate transcription.

A promoter may be one naturally associated with a gene or polynucleotidesequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a polynucleotide sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding polynucleotidesegment under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with apolynucleotide sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a polynucleotide sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR, inconnection with the compositions disclosed herein (U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906). Furthermore, it is contemplated thecontrol sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know how to use promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (2012). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or polypeptides. The promoter may beheterologous or endogenous.

One example of a constitutive promoter sequence is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.However, other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, Moloney virus promoter, the avianleukemia virus promoter, Epstein-Barr virus immediate early promoter,Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the muscle creatine promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter in the invention provides a molecularswitch capable of turning on expression of the polynucleotide sequencewhich it is operatively linked when such expression is desired, orturning off the expression when expression is not desired. Examples ofinducible promoters include, but are not limited to a metallothioninepromoter, a glucocorticoid promoter, a progesterone promoter, and atetracycline promoter. Further, the invention includes the use of atissue-specific promoter, where the promoter is active only in a desiredtissue. Tissue-specific promoters are well known in the art and include,but are not limited to, the HER-2 promoter and the PSA associatedpromoter sequences.

In order to assess the expression of the nucleotide sequences encodingthe polypeptide or combinations of polypeptides of the invention, theexpression vector to be introduced into a cell can also contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother embodiments, the selectable marker may be carried on a separatepiece of DNA and used in a co-transfection procedure. Both selectablemarkers and reporter genes may be flanked with appropriate regulatorysequences to enable expression in the host cells. Useful selectablemarkers are known in the art and include, for example,antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells.

Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (see, e.g.,Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systemsare well known and may be prepared using well known techniques orobtained commercially. Internal deletion constructs may be generatedusing unique internal restriction sites or by partial digestion ofnon-unique restriction sites. Constructs may then be transfected intocells that display high levels of siRNA polynucleotide and/orpolypeptide expression. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In some embodiments, the expression vector is modified to increase theexpression of the desired polypeptide. For example, the vector canundergo codon optimization to improve expression in a given mammal. Forexample, the vector can be codon-optimized for human expression. Inanother embodiment, the expression vector comprises an effectivesecretory leader. An exemplary leader is an IgE leader sequence. Inanother embodiment, the expression vector comprises a Kozak element toinitiate translation. In another embodiment, the nucleic acid is removedof cis-acting sequence motifs/RNA secondary structures that would impedetranslation. Such modifications, and others, are known in the art foruse in DNA vaccines (Kutzler et al, 2008, Nat. Rev. Gen. 9: 776-788; PCTApp. No. PCT/US2007/000886; PCT App. No.; PCT/US2004/018962).

Vaccine

For an antigenic composition to be useful as a vaccine, the antigeniccomposition must induce an immune response to the antigen in a cell,tissue or subject (e.g., a human). Preferably, the vaccine induces aprotective immune response in the subject. As used herein, an“immunological composition” may comprise, by way of examples, an antigen(e.g., a polypeptide), a nucleic acid encoding an antigen (e.g., anantigen expression vector), or a cell expressing or presenting anantigen. In particular embodiments the antigenic composition comprisesor encodes all or part of any polypeptide antigen described herein, oran immunologically functional equivalent thereof. In other embodiments,the antigenic composition is in a mixture that comprises an additionalimmunostimulatory agent or nucleic acids encoding such an agent.Immunostimulatory agents include but are not limited to an additionalantigen, an immunomodulator, an antigen presenting cell or an adjuvant.In other embodiments, one or more of the additional agent(s) iscovalently bonded to the antigen or an immunostimulatory agent, in anycombination. In certain embodiments, the antigenic composition isconjugated to or comprises an HLA anchor motif amino acids.

In the context of the present invention, the term “vaccine” (alsoreferred to as an immunogenic composition) refers to a substance thatinduces anti-S. typhi or S. paratyphi immunity or suppresses S. typhi orS. paratyphi upon inoculation into an animal. In various embodiments,the vaccine of the invention comprises at least one of PltA, PltB, CdtB,or mutants thereof, of S. typhi or and S. paratyphi to a subject toinduce an immune response. In one embodiment, the vaccine isadministered in combination with an adjuvant. In another embodiment, thevaccine is administered in the absence of an adjuvant.

A vaccine of the present invention may vary in its composition ofnucleic acid and/or cellular components. In a non-limiting example, anucleic encoding an antigen might also be formulated with an adjuvant.Of course, it will be understood that various compositions describedherein may further comprise additional components. For example, one ormore vaccine components may be comprised in a lipid or liposome. Inanother non-limiting example, a vaccine may comprise one or moreadjuvants. A vaccine of the present invention, and its variouscomponents, may be prepared and/or administered by any method disclosedherein or as would be known to one of ordinary skill in the art, inlight of the present disclosure.

In one embodiment, the polypeptide vaccine of the invention includes,but is not limited to at least one polypeptide, or a fragment thereof,optionally mixed with adjuvant substances. In some embodiments, thepolypeptide is introduced together with an antigen presenting cell(APC). The most common cells used for the latter type of vaccine arebone marrow and peripheral blood derived dendritic cells, as these cellsexpress costimulatory molecules that help activation of T cells.WO00/06723 discloses a cellular vaccine composition which includes anAPC presenting tumor associated antigen polypeptides. Presenting thepolypeptide can be effected by loading the APC with a polynucleotide(e.g., DNA, RNA) encoding the polypeptide or loading the APC with thepolypeptide itself.

Thus, the present invention also encompasses a method of inducing S.typhi or S. paratyphi immunity using one or more of polypeptidesdescribed herein. When a certain polypeptide or combination ofpolypeptides induces an S. typhi or S. paratyphi immune response uponinoculation into an animal, the polypeptide or combination ofpolypeptides are determined to have an immunity inducing effect. Theinduction of the S. typhi or S. paratyphi immunity by a polypeptide orcombination of polypeptides can be detected by observing in vivo or invitro the response of the immune system in the host against thepolypeptide.

In another embodiment, the methods of the invention compriseadministering to the subject a bacterium or virus comprising a nucleicacid sequence encoding at least one of PltA, PltB, CdtB, or mutantsthereof. In another embodiment, the methods of the invention compriseadministering to the subject a bacterium or virus expressing at least aportion of at least one of PltA, PltB, CdtB, or mutants thereof. Inanother embodiment, the methods of the invention comprise administeringto the subject a bacterium or virus comprising at least a portion of atleast one of PltA, PltB, CdtB, or mutants thereof.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. A foreign substance that enters the livingbody is presented to T cells and B cells by the action of APCs. T cellsthat respond to the antigen presented by APC in an antigen-specificmanner differentiate into cytotoxic T cells (also referred to ascytotoxic T lymphocytes or CTLs) due to stimulation by the antigen.These antigen stimulated cells then proliferate. This process isreferred to herein as “activation” of T cells. Therefore, CTL inductionby a certain polypeptide or combination of polypeptides of the inventioncan be evaluated by presenting the polypeptide to a T cell by APC, anddetecting the induction of CTL. Furthermore, APCs have the effect ofactivating CD4+T cells, CD8+T cells, macrophages, eosinophils and NKcells.

A method for evaluating the inducing action of CTL using dendritic cells(DCs) as APC is well known in the art. DC is a representative APC havingthe strongest CTL inducing action among APCs. In this method, thepolypeptide or combination of polypeptides are initially contacted withDC and then this DC is contacted with T cells. Detection of T cellshaving cytotoxic effects against the cells of interest after the contactwith DC shows that the polypeptide or combination of polypeptides havean activity of inducing the cytotoxic T cells. Furthermore, the inducedimmune response can be also examined by measuring IFN-gamma produced andreleased by CTL in the presence of antigen-presenting cells that carryimmobilized polypeptide or combination of polypeptides by visualizingusing anti-IFN-gamma antibodies, such as an ELISPOT assay.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also beused as the APC. The induction of CTL is reported to be enhanced byculturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL hasbeen shown to be induced by culturing PBMC in the presence of keyholelimpet hemocyanin (KLH) and IL-7.

The polypeptide, or combination of polypeptides, confirmed to possessCTL inducing activity by these methods are polypeptides having DCactivation effect and subsequent CTL inducing activity. Therefore, apolypeptide or combination of polypeptides that induce CTL against toxinA and toxin B are useful as vaccines against S. typhi or S. paratyphiassociated pathology. Furthermore, CTL that have acquired cytotoxicitydue to presentation of the polypeptide or combination of polypeptides byAPC can be also used as vaccines against S. typhi or S. paratyphiinfection.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction can be increased by combining aplurality of polypeptides having different structures and contactingthem with DC. Therefore, when stimulating DC with protein fragments, itis advantageous to use a mixture of multiple types of fragments.

The induction of S. typhi or S. paratyphi immunity by a polypeptide orcombination of polypeptides can be further confirmed by observing theinduction of antibody production against the specific toxins. Forexample, when antibodies against a polypeptide or combination ofpolypeptides are induced in a laboratory animal immunized with thepolypeptide or combination of polypeptides, and when S. typhi or S.paratyphi associated pathology is suppressed by those antibodies, thepolypeptide or combination of polypeptides are determined to induceanti-S. typhi or anti-S. paratyphi immunity.

S. typhi or S. paratyphi immunity can be induced by administering avaccine of the invention, and the induction of S. typhi or S. paratyphiimmunity enables treatment and prevention of pathologies associated withS. typhi or S. paratyphi. Thus, the invention provides a method fortreating, or preventing infection by S. typhi or S. paratyphi. Thetherapeutic compounds or compositions of the invention may beadministered prophylactically or therapeutically to subjects sufferingfrom, or at risk of, or susceptible to, developing S. typhi or S.paratyphi infection. Such subjects may be identified using standardclinical methods. In the context of the present invention, prophylacticadministration occurs prior to the manifestation of overt clinicalsymptoms of disease, such that a disease or disorder is prevented oralternatively delayed in its progression. In the context of the field ofmedicine, the term “prevent” encompasses any activity which reduces theburden of mortality or morbidity from disease. Prevention can occur atprimary, secondary and tertiary prevention levels. While primaryprevention avoids the development of a disease, secondary and tertiarylevels of prevention encompass activities aimed at preventing theprogression of a disease and the emergence of symptoms as well asreducing the negative impact of an already established disease byrestoring function and reducing disease-related complications.

The polypeptide or combination of polypeptides of the invention havingimmunological activity, or a polynucleotide or vector encoding such apolypeptide or combination of polypeptides, may optionally be combinedwith an adjuvant. An adjuvant refers to a compound that enhances theimmune response against the polypeptide or combination of polypeptideswhen administered together (or successively) with the polypeptide havingimmunological activity. Examples of suitable adjuvants include choleratoxin, salmonella toxin, alum and such, but are not limited thereto.Furthermore, a vaccine of this invention may be combined appropriatelywith a pharmaceutically acceptable carrier. Examples of such carriersare sterilized water, physiological saline, phosphate buffer, culturefluid and such. Furthermore, the vaccine may contain as necessary,stabilizers, suspensions, preservatives, surfactants and such. Thevaccine is administered systemically or locally. Vaccine administrationmay be performed by single administration or boosted by multipleadministrations.

Administration

In one embodiment, the methods of the present invention compriseadministering a composition comprising at least one polypeptide of theinvention, and/or at least one polynucleotide encoding at least onepolypeptide of the invention, to a subject. Administration of thecomposition can comprise, for example, intramuscular, intravenous,peritoneal, subcutaneous, intradermal, as well as topicaladministration.

The actual dose and schedule can vary depending on whether thecompositions are administered in combination with other pharmaceuticalcompositions, or depending on inter-individual differences inpharmacokinetics, drug disposition, and metabolism. Similarly, amountscan vary in in vitro applications depending on the particular cell lineutilized (e.g., based on the number of vector receptors present on thecell surface, or the ability of the particular vector employed for genetransfer to replicate in that cell line). Furthermore, the amount ofvector to be added per cell will likely vary with the length andstability of the therapeutic gene inserted in the vector, as well asalso the nature of the sequence, and is particularly a parameter whichneeds to be determined empirically, and can be altered due to factorsnot inherent to the methods of the present invention (for instance, thecost associated with synthesis). One skilled in the art can easily makeany necessary adjustments in accordance with the exigencies of theparticular situation.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Therapeutic Inhibitor Compositions and Methods

In various embodiments, the present invention includes inhibitorcompositions for inhibiting with the interaction between the S. typhi orS. paratyphi toxin and the toxin's receptor. Inhibition of theinteraction between the S. typhi or S. paratyphi toxin and the toxin'sreceptor can be assessed using a wide variety of methods, includingthose disclosed herein, as well as methods known in the art or to bedeveloped in the future.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the inhibitor compositions and methods of the inventionare useful in treating preventing and infection by S. typhi and S.paratyphi.

The inhibitor compositions and methods of the invention that interferewith the interaction between the S. typhi or S. paratyphi toxin and thetoxin's receptor include, but should not be construed as being limitedto, a chemical compound, a protein, a peptide, a peptidomemetic, anantibody, a ribozyme, a small molecule chemical compound, a glycan, anantisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), orcombinations thereof. One of skill in the art would readily appreciate,based on the disclosure provided herein, that the inhibitor compositionsof the invention include those that interfere with the interactionbetween the toxin and its receptor. In some embodiments, the inhibitorcompositions bind to the toxin and interfere with the interactionbetween the toxin and its receptor. In other embodiments, the inhibitorcompositions bind to the toxin's receptor and interfere with theinteraction between the toxin and its receptor.

In various embodiments, the treatment of S. typhi or S. paratyphiinfection in a subject is accomplished through passive antibody therapy(i.e., the transfer of antibodies to the S. typhi or S. paratyphiinfected subject). In various embodiments, the inhibitor compositionsand methods of the invention that interfere with the interaction betweenthe S. typhi or S. paratyphi toxin and the toxin's receptor are used incombination with an antibiotic therapy. When used in combination, theantibiotic therapy can be administered before, during or after theadministration of the inhibitor compositions of the invention.

In some embodiments, the receptor for the S. typhi or S. paratyphi toxinis a glycan. In one embodiment, the glycan is an N-linked glycan suchas, by way of non-limiting examples, a sialylated tri- or bi-antennaryglycan with one or all of the branches terminally sialyated. In anotherembodiment, the glycan is a non-sialylated tri- or bi-antennary glycan.In another embodiment, the glycan is a ganglioside. In anotherembodiment, the glycan is a glycan found on O-glycans. In variousembodiments, the glycan is any one or more of the glycans listed inFIGS. 20, 21 and 22.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that aninhibitor composition includes such inhibitors as discovered in thefuture, as can be identified by well-known criteria in the art ofpharmacology, such as the physiological results of inhibition of thetoxin as described in detail herein and/or as known in the art.Therefore, the present invention is not limited in any way to anyparticular inhibitor composition as exemplified or disclosed herein;rather, the invention encompasses those inhibitor compositions thatwould be understood by the routineer to be useful as are known in theart and as are discovered in the future.

Further methods of identifying and producing inhibitor compositions arewell known to those of ordinary skill in the art, including, but notlimited, obtaining an inhibitor from a naturally occurring source (i.e.,Streptomyces sp., Pseudomonas sp., Stylotella aurantium). Alternatively,an inhibitor can be synthesized chemically. Further, the routineer wouldappreciate, based upon the teachings provided herein, that an inhibitorcomposition can be obtained from a recombinant organism. Compositionsand methods for chemically synthesizing inhibitors and for obtainingthem from natural sources are well known in the art and are described inthe art.

One of skill in the art will appreciate that an inhibitor can beadministered as a small molecule chemical, a protein, an antibody, aglycan, a nucleic acid construct encoding a protein, an antisensenucleic acid, a nucleic acid construct encoding an antisense nucleicacid, or combinations thereof. In one embodiment, the inhibitorcomposition of the invention that interferes with the interactionbetween the S. typhi or S. paratyphi toxin and the toxin's receptor is asoluble form of at least a fragment of at least one glycan that is areceptor for the S. typhi or S. paratyphi toxin. In various embodiments,the soluble form of at least a fragment of at least one glycan is asoluble form of at least a fragment of at least one glycan listed inFIGS. 20, 21 and 22.

Numerous vectors and other compositions and methods are well known foradministering a protein or a nucleic acid construct encoding a proteinto cells or tissues. Therefore, the invention includes a method ofadministering a protein or a nucleic acid encoding a protein that is aninhibitor. (Sambrook et al., 2012, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York).

One of skill in the art will appreciate that inhibitors of the inventioncan be administered singly or in any combination. Further, inhibitorscan be administered singly or in any combination in a temporal sense, inthat they may be administered concurrently, or before, and/or after eachother.

In various embodiments, any of the inhibitors of the invention describedherein can be administered alone or in combination with other inhibitorsof other molecules associated with S. typhi or S. paratyphi infection.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder that isalready established. Particularly, the disease or disorder need not havemanifested to the point of detriment to the subject; indeed, the diseaseor disorder need not be detected in a subject before treatment isadministered. That is, significant disease or disorder does not have tooccur before the present invention may provide benefit. Therefore, thepresent invention includes a method for preventing infection in asubject, in that an inhibitor composition, as discussed previouslyelsewhere herein, can be administered to a subject prior to the onset ofthe infection.

The invention encompasses administration of an inhibitor to practice themethods of the invention; the skilled artisan would understand, based onthe disclosure provided herein, how to formulate and administer theappropriate inhibitor to a subject. Indeed, the successfuladministration of the inhibitor has been reduced to practice herein.However, the present invention is not limited to any particular methodof administration or treatment regimen.

Pharmaceutical Compositions

The present invention includes the treatment of a S. typhi or S.paratyphi infection in a subject by the administration of a therapeuticcomposition of the invention to a subject in need thereof. In oneembodiment, the therapeutic composition of the invention is an inhibitorcomposition. In one embodiment, the therapeutic composition of theinvention for the treatment of S. typhi or S. paratyphi infection is atleast one antibody that specifically binds to at least one of PltA,PltB, CdtB, or mutants thereof. In various embodiments, the treatment ofS. typhi or S. paratyphi infection in a subject is accomplished throughpassive antibody therapy (i.e., the transfer of antibodies to the S.typhi or S. paratyphi infected subject).

Administration of the therapeutic composition in accordance with thepresent invention may be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of thecompositions of the invention may be essentially continuous over apreselected period of time or may be in a series of spaced doses. Bothlocal and systemic administration is contemplated. The amountadministered will vary depending on various factors including, but notlimited to, the composition chosen, the particular disease, the weight,the physical condition, and the age of the subject, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems which are well known to the art.

When the compositions of the invention are prepared for administration,they are preferably combined with a pharmaceutically acceptable carrier,diluent or excipient to form a pharmaceutical formulation, or unitdosage form. The total active ingredients in such formulations includefrom 0.1 to 99.9% by weight of the formulation. A “pharmaceuticallyacceptable” is a carrier, diluent, excipient, and/or salt that iscompatible with the other ingredients of the formulation, and notdeleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules; as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the compositions of the inventioncan be prepared by procedures known in the art using well known andreadily available ingredients. The compositions of the invention canalso be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

Thus, the composition may be formulated for parenteral administration(e.g., by injection, for example, bolus injection or continuousinfusion) and may be presented in unit dose form in ampules, pre-filledsyringes, small volume infusion containers or in multi-dose containerswith an added preservative. The active ingredients may take such formsas suspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the active ingredients may be inpowder form, obtained by aseptic isolation of sterile solid or bylyophilization from solution, for constitution with a suitable vehicle,e.g., sterile, pyrogen-free water, before use.

In general, water, suitable oil, saline, aqueous dextrose (glucose), andrelated sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration contain the active ingredient,suitable stabilizing agents and, if necessary, buffer substances.Antioxidizing agents such as sodium bisulfate, sodium sulfite orascorbic acid, either alone or combined, are suitable stabilizingagents. Also used are citric acid and its salts and sodiumethylenediaminetetraacetic acid (EDTA). In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, astandard reference text in this field.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Methods of Diagnosis

In other embodiments, the invention is a method of determining whether asubject is, or has been, infected with S. typhi or S. paratyphi. In someembodiments, the method comprises detecting or measuring the level oftyphoid toxin in the subject. In various embodiments, the methodcomprises detecting or measuring the level of at least one of PltA, CdtBand PltB in the subject. In some embodiments, the method comprisesdetecting or measuring the level of antibodies that specifically bind tothe typhoid toxin in the subject. In various embodiments, the methodcomprises detecting or measuring the level of at least one antibody thatspecifically binds to PltA, CdtB or PltB in the subject.

In one embodiment, the invention is a method of determining whether asubject is infected with S. typhi or S. paratyphi, comprising the stepof detecting or measuring the level of typhoid toxin in a biologicalsample of the subject. In various embodiments, the method comprisesdetecting or measuring the level of typhoid toxin by detecting ormeasuring the level of at least one of PltA, CdtB and PltB in thebiological sample of the subject. In various embodiments, to determinewhether the level of typhoid toxin is elevated in a biological sample ofthe subject, the level of typhoid toxin is compared with the level of atleast one comparator control, such as a positive control, a negativecontrol, a historical control, a historical norm, or the level ofanother reference molecule in the biological sample. The results of thediagnostic assay can be used alone, or in combination with otherinformation from the subject, or other information from the biologicalsample obtained from the subject.

In one embodiment, the invention is a method of determining whether asubject is, or has been, infected with S. typhi or S. paratyphi,comprising the step of detecting or measuring the level of antibodiesthat specifically bind to the typhoid toxin in a biological sample ofthe subject. In various embodiments, the method comprises detecting ormeasuring the level of antibodies that specifically bind to typhoidtoxin by detecting or measuring the level of at least one antibody thatspecifically binds to PltA, CdtB, or PltB in the biological sample ofthe subject. In various embodiments, to determine whether the level ofantibodies that specifically bind to typhoid toxin is elevated in abiological sample of the subject, the level of antibodies thatspecifically bind to typhoid toxin is compared with the level of atleast one comparator control, such as a positive control, a negativecontrol, a historical control, a historical norm, or the level ofanother reference molecule in the biological sample. The results of thediagnostic assay can be used alone, or in combination with otherinformation from the subject, or other information from the biologicalsample obtained from the subject.

In various embodiments of the methods of the invention, the level ofantibody that specifically binds to typhoid toxin is determined to beelevated when the level of antibody that specifically binds to typhoidtoxin is increased by at least 1%, at least 5%, at least 10%, by atleast 20%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 80%, by at least 90%, by atleast 100%, by at least 125%, by at least 150%, by at least 175%, by atleast 200%, by at least 250%, by at least 300%, by at least 400%, by atleast 500%, by at least 600%, by at least 700%, by at least 800%, by atleast 900%, by at least 1000%, by at least 1500%, by at least 2000%, byat least 2500%, by at least 3000%, by at least 4000%, or by at least5000%, when compared with a comparator control.

In various embodiments, the biological sample is a sample containing apolypeptide or nucleic acid of at least one of PltA, CdtB, PltB, anantibody the specifically binds to PltA, an antibody the specificallybinds to CdtB, or an antibody the specifically binds to PltB. Thebiological sample can be a sample from any source which contains apolypeptide or a nucleic acid, such as a bodily fluid or a tissue, or acombination thereof. A biological sample can be obtained by appropriatemethods, such as, by way of examples, blood draw, fluid draw, or biopsy.A biological sample can be used as the test sample; alternatively, abiological sample can be processed to enhance access to the polypeptidesor nucleic acids, or copies of the nucleic acids, and the processedbiological sample can then be used as a test sample.

In various embodiments of the invention, methods of detecting ormeasuring the level of at least one of PltA, CdtB, PltB, an antibody thespecifically binds to PltA, an antibody the specifically binds to CdtB,or an antibody the specifically binds to PltB levels in a biologicalsample obtained from a patient include, but are not limited to, animmunochromatography assay, an immunodot assay, a Luminex assay, anELISA assay, an ELISPOT assay, a protein microarray assay, aligand-receptor binding assay, displacement of a ligand from a receptorassay, displacement of a ligand from a shared receptor assay, animmunostaining assay, a Western blot assay, a mass spectrophotometryassay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquidchromatography-tandem mass spectrometry assay, an ouchterlonyimmunodiffusion assay, reverse phase protein microarray, a rocketimmunoelectrophoresis assay, an immunohistostaining assay, animmunoprecipitation assay, a complement fixation assay, FACS, anenzyme-substrate binding assay, an enzymatic assay, an enzymatic assayemploying a detectable molecule, such as a chromophore, fluorophore, orradioactive substrate, a substrate binding assay employing such asubstrate, a substrate displacement assay employing such a substrate,and a protein chip assay (see also, 2007, Van Emon, Immunoassay andOther Bioanalytical Techniques, CRC Press; 2005, Wild, ImmunoassayHandbook, Gulf Professional Publishing; 1996, Diamandis andChristopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays inClinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, ProteomicsToday, John Wiley and Sons; 2007).

In various embodiments of the invention, methods of detecting ormeasuring the level of at least one of PltA, CdtB, PltB, an antibody thespecifically binds to PltA, an antibody the specifically binds to CdtB,or an antibody the specifically binds to PltB levels in a biologicalsample obtained from a patient include, but are not limited to,quantitative hybridization methods, such as Southern analysis, Northernanalysis, or in situ hybridizations, (see Current Protocols in MolecularBiology, Ausubel, F. et al., eds., John Wiley & Sons, including allsupplements). A “nucleic acid probe,” as used herein, can be a DNA probeor an RNA probe. The probe can be, for example, a gene, a gene fragment(e.g., one or more exons), a vector comprising the gene, a probe orprimer, etc. For representative examples of use of nucleic acid probes,see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330. The nucleicacid probe can be, for example, a full-length nucleic acid molecule, ora portion thereof, such as an oligonucleotide of at least 15, 30, 50,100, 250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to appropriate target mRNA or cDNA.The hybridization sample is maintained under conditions which aresufficient to allow specific hybridization of the nucleic acid probe tomRNA or cDNA. Specific hybridization can be performed under highstringency conditions or moderate stringency conditions, as appropriate.In a preferred embodiment, the hybridization conditions for specifichybridization are high stringency. Specific hybridization, if present,is then detected using standard methods. If specific hybridizationoccurs between the nucleic acid probe having a mRNA or cDNA in the testsample, the level of the mRNA or cDNA in the sample can be assessed.More than one nucleic acid probe can also be used concurrently in thismethod. Specific hybridization of any one of the nucleic acid probes isindicative of the presence of the mRNA or cDNA of interest, as describedherein.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the quantitative hybridization methods describedherein. PNA is a DNA mimic having a peptide-like, inorganic backbone,such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, Tor U) attached to the glycine nitrogen via a methylene carbonyl linker(see, for example, 1994, Nielsen et al., Bioconjugate Chemistry 5:1).The PNA probe can be designed to specifically hybridize to a targetnucleic acid sequence. Hybridization of the PNA probe to a nucleic acidsequence is used to determine the level of the target nucleic acid inthe biological sample.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target nucleic acid sequences in the biological sampleobtained from a subject can be used to determine the level of typhoidtoxin in the biological sample of a subject. The array ofoligonucleotide probes can be used to determine the level of typhoidtoxin, or at least one of PltA, CdtB, PltB, alone, or the level oftyphoid toxin, or at least one of PltA, CdtB, PltB, in relation to thelevel of one or more other nucleic acids in the biological sample.Oligonucleotide arrays typically comprise a plurality of differentoligonucleotide probes that are coupled to a surface of a substrate indifferent known locations. These oligonucleotide arrays, also known as“Genechips,” have been generally described in the art, for example, U.S.Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and92/10092. These arrays can generally be produced using mechanicalsynthesis methods or light directed synthesis methods which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis methods. See Fodor et al., Science,251:767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCT Publication No.WO 92/10092 and U.S. Pat. No. 5,424,186. Techniques for the synthesis ofthese arrays using mechanical synthesis methods are described in, e.g.,U.S. Pat. No. 5,384,261.

After an oligonucleotide array is prepared, a nucleic acid of interestis hybridized with the array and its level is quantified. Hybridizationand quantification are generally carried out by methods described hereinand also in, e.g., published PCT Application Nos. WO 92/10092 and WO95/11995, and U.S. Pat. No. 5,424,186. In brief, a target nucleic acidsequence is amplified by well-known amplification techniques, e.g., PCR.Typically, this involves the use of primer sequences that arecomplementary to the target nucleic acid. Asymmetric PCR techniques mayalso be used. Amplified target, generally incorporating a label, is thenhybridized with the array under appropriate conditions. Upon completionof hybridization and washing of the array, the array is scanned todetermine the quantity of hybridized nucleic acid. The hybridizationdata obtained from the scan is typically in the form of fluorescenceintensities as a function of quantity, or relative quantity, of thetarget nucleic acid in the biological sample. The target nucleic acidcan be hybridized to the array in combination with one or morecomparator controls (e.g., positive control, negative control, quantitycontrol, etc.) to improve quantification of the target nucleic acid inthe sample.

The probes and primers according to the invention can be labeleddirectly or indirectly with a radioactive or nonradioactive compound, bymethods well known to those skilled in the art, in order to obtain adetectable and/or quantifiable signal; the labeling of the primers or ofthe probes according to the invention is carried out with radioactiveelements or with nonradioactive molecules. Among the radioactiveisotopes used, mention may be made of 32P, 33P, 35S or 3H. Thenonradioactive entities are selected from ligands such as biotin,avidin, streptavidin or digoxigenin, haptenes, dyes, and luminescentagents such as radioluminescent, chemoluminescent, bioluminescent,fluorescent or phosphorescent agents.

Nucleic acids can be obtained from the cells using known techniques.Nucleic acid herein refers to RNA, including mRNA, and DNA, includingcDNA. The nucleic acid can be double-stranded or single-stranded (i.e.,a sense or an antisense single strand) and can be complementary to anucleic acid encoding a polypeptide. The nucleic acid content may alsobe an RNA or DNA extraction performed on a biological sample, includinga biological fluid and fresh or fixed tissue sample.

There are many methods known in the art for the detection andquantification of specific nucleic acid sequences and new methods arecontinually reported. A great majority of the known specific nucleicacid detection and quantification methods utilize nucleic acid probes inspecific hybridization reactions. Preferably, the detection ofhybridization to the duplex form is a Southern blot technique. In theSouthern blot technique, a nucleic acid sample is separated in anagarose gel based on size (molecular weight) and affixed to a membrane,denatured, and exposed to (admixed with) the labeled nucleic acid probeunder hybridizing conditions. If the labeled nucleic acid probe forms ahybrid with the nucleic acid on the blot, the label is bound to themembrane.

In the Southern blot, the nucleic acid probe is preferably labeled witha tag. That tag can be a radioactive isotope, a fluorescent dye or theother well-known materials. Another type of process for the specificdetection of nucleic acids in a biological sample known in the art arethe hybridization methods as exemplified by U.S. Pat. No. 6,159,693 andNo. 6,270,974, and related patents. To briefly summarize one of thosemethods, a nucleic acid probe of at least 10 nucleotides, preferably atleast 15 nucleotides, more preferably at least 25 nucleotides, having asequence complementary to a nucleic acid of interest is hybridized in asample, subjected to depolymerizing conditions, and the sample istreated with an ATP/luciferase system, which will luminesce if thenucleic sequence is present. In quantitative Southern blotting, thelevel of the nucleic acid of interest can be compared with the level ofa second nucleic acid of interest, and/or to one or more comparatorcontrol nucleic acids (e.g., positive control, negative control,quantity control, etc.).

Many methods useful for the detection and quantification of nucleic acidtakes advantage of the polymerase chain reaction (PCR). The PCR processis well known in the art (U.S. Pat. No. 4,683,195, No. 4,683,202, andNo. 4,800,159). To briefly summarize PCR, nucleic acid primers,complementary to opposite strands of a nucleic acid amplification targetsequence, are permitted to anneal to the denatured sample. A DNApolymerase (typically heat stable) extends the DNA duplex from thehybridized primer. The process is repeated to amplify the nucleic acidtarget. If the nucleic acid primers do not hybridize to the sample, thenthere is no corresponding amplified PCR product. In this case, the PCRprimer acts as a hybridization probe.

In PCR, the nucleic acid probe can be labeled with a tag as discussedelsewhere herein. Most preferably the detection of the duplex is doneusing at least one primer directed to the nucleic acid of interest. Inyet another embodiment of PCR, the detection of the hybridized duplexcomprises electrophoretic gel separation followed by dye-basedvisualization.

Typical hybridization and washing stringency conditions depend in parton the size (i.e., number of nucleotides in length) of theoligonucleotide probe, the base composition and monovalent and divalentcation concentrations (Ausubel et al., 1994, eds Current Protocols inMolecular Biology).

In a preferred embodiment, the process for determining the quantitativeand qualitative profile of the nucleic acid of interest according to thepresent invention is characterized in that the amplifications arereal-time amplifications performed using a labeled probe, preferably alabeled hydrolysis-probe, capable of specifically hybridizing instringent conditions with a segment of the nucleic acid of interest. Thelabeled probe is capable of emitting a detectable signal every time eachamplification cycle occurs, allowing the signal obtained for each cycleto be measured.

The real-time amplification, such as real-time PCR, is well known in theart, and the various known techniques will be employed in the best wayfor the implementation of the present process. These techniques areperformed using various categories of probes, such as hydrolysis probes,hybridization adjacent probes, or molecular beacons. The techniquesemploying hydrolysis probes or molecular beacons are based on the use ofa fluorescence quencher/reporter system, and the hybridization adjacentprobes are based on the use of fluorescence acceptor/donor molecules.

Hydrolysis probes with a fluorescence quencher/reporter system areavailable in the market, and are for example commercialized by theApplied Biosystems group (USA). Many fluorescent dyes may be employed,such as FAM dyes (6-carboxy-fluorescein), or any other dyephosphoramidite reagents.

Among the stringent conditions applied for any one of thehydrolysis-probes of the present invention is the Tm, which is in therange of about 65° C. to 75° C. Preferably, the Tm for any one of thehydrolysis-probes of the present invention is in the range of about 67°C. to about 70° C. Most preferably, the Tm applied for any one of thehydrolysis-probes of the present invention is about 67° C.

In one aspect, the invention includes a primer that is complementary toa nucleic acid of interest, and more particularly the primer includes 12or more contiguous nucleotides substantially complementary to thenucleic acid of interest. Preferably, a primer featured in the inventionincludes a nucleotide sequence sufficiently complementary to hybridizeto a nucleic acid sequence of about 12 to 25 nucleotides. Morepreferably, the primer differs by no more than 1, 2, or 3 nucleotidesfrom the target flanking nucleotide sequence In another aspect, thelength of the primer can vary in length, preferably about 15 to 28nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,or 27 nucleotides in length).

EXPERIMENTAL EXAMPLE

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1 Conferring Virulence: Structure and Function of the ChimericA₂B₅ Typhoid Toxin

The results described herein demonstrate that the systemicadministration of typhoid toxin, a unique virulence factor of S. typhi,reproduces many of the acute symptoms of typhoid fever. Specificcarbohydrate moieties on specific surface glycoproteins that serve asreceptors for typhoid toxin were identified. These carbohydrate moietiesprovide the broad cell target specificity of typhoid toxin. The atomicstructure of typhoid toxin was identified, which shows an unprecedentedA₂B₅ organization with two covalently-linked A subunitsnon-covalently-associated to a pentameric B subunit. The structureprovides insight into the toxin's receptor-binding specificity anddelivery mechanisms. The structure also demonstrates how the activitiesof two powerful toxins have been co-opted into a single, unique toxinthat can induce many of the symptoms characteristic of typhoid fever.These findings are useful for the life-saving therapeutics againsttyphoid fever.

The materials and methods employed in these experiments are nowdescribed.

Bacterial Strains, Mammalian Cells, and Growth Conditions

The wild-type Salmonella enterica serovar Typhi strain ISP2825 has beendescribed previously (Galan and Curtiss, 1991, Infect. Immun.59:2901-2908). A derivative of this strain encoding a FLAG-epitopetagged CdtB has been previously described (Spano et al., 2008, Cell HostMicrobe 3:30-38). Other S. typhi mutant strains were constructed byallelic exchange using previously described methods (Kaniga et al.,1994, Mol. Microbiol. 13:555-568). Site directed mutations and epitopetagging was carried out following standard recombinant DNA procedures.

For all infection experiments, S. typhi strains were grown at 37° C. in2 ml LB broth containing 0.3 M NaCl to an OD600 of ˜0.9 afterinoculation from overnight cultures at a dilution of 1:50. Culturedcells and culture medium used in these studies were as follows:

Henle-407 (human intestinal epithelial cells): DMEM+10% BCS;

Jurkat (human T lymphocytes): RPMI1640+10% FBS;

Ramos (human B lymphocytes): RPMI1640+10% FBS;

THP1 (human monocytic cells): RPMI1640+10% FBS+0.05 mMβ-mercaptoethanol;

Raw (mouse monocytes/macrophages): DMEM+10% FBS;

NIH3T3 (mouse embryonic fibroblasts): DMEM+10% BCS;

COS1 (monkey kidney fibroblasts): DMEM+10% BCS;

CHO (Chinese hamster ovary epithelial cells): Ham's F12+10% FBS;

MDCK (canine kidney epithelial cells): MEM+10% FBS;

Lec-1 (N-acetylglucosaminyltransferase I mutant) and parent cell Pro-5:alpha MEM with ribonucleosides and deoxyribonucleosides+10% FBS.

All mammalian cells were cultured at 37° C. under an atmosphere of 5%CO₂.

Typhoid Toxin Expression and Purification

PltB, PltA, and CdtB (wild type or mutant alleles, and 3×FLAG or 6×Histagged at the carboxy terminus of CdtB, as indicated) were cloned as asingle operon in either a low copy plasmid derived from pWSK129 (Wangand Kushner, 1001, Gene 100:195-199), or in pET28a+(Novagen). E. colistrains carrying the different plasmids were grown to an OD600 of0.6-0.7 at 37° C., expression of typhoid toxin was subsequently inducedby the addition of 0.5 mM IPTG, and induced cultures were incubatedovernight at 30° C. Bacterial cells were pelleted by centrifugation, andbacterial cells were resuspended in a buffer containing 200 mM Tris-HCl(pH 7.5), 20% sucrose, 1 mM EDTA, 2 mg/ml lysozyme, and lx proteaseinhibitors (2 ml per each gram of wet cell pellet weight), incubated for5 min at RT, mixed with dH₂O (3 ml per each gram of wet cell pelletweight) by inversion, and incubated for additional 10 min on ice. Acrude periplasmic protein fraction was obtained by centrifugation at4,000×g for 15 min at RT, and used as a source of typhoid toxin foraffinity chromatography purification using M2 Flag affinity gel (Sigma).The periplasmic fraction containing typhoid holotoxin was incubated for3 hrs at 4° C. with M2 agarose beads packed on a 10 ml column (Bio-rad),washed with ˜20 bed volume of PBS, and eluted twice with PBS containing3×Flag peptide (Sigma; 150 ng/μl). Partially purified holotoxin wasapplied on a superdex 200 size-exclusion chromatography (Tris-HClbuffer, 15-50 mM, in a pH range of 7.6-8.0 supplemented with 150 mMNaCl) to complete its purification.

His-epitope-tagged typhoid toxin was purified as follows. E. colicultures were resuspended in a buffer containing 15 mM Tris-HCl, pH8.0,150 mM NaCl, 2 mg/ml lysozyme, 10 μg/ml DNase, and 1×protease inhibitorcocktail, lysed by passing them through a French press three times,pelleted, and affinity-purified using a Nickel-resin (Qiagen) accordingto the vendor's recommendation. The eluates were diluted in 20 mM MES,pH6.0 buffer and loaded onto a Hitrap ion-exchange column. Fractionsfrom the ion-exchange chromatography were monitored on SDS-PAGE,concentrated, and further purified by using a superdex 200 column. Finalfractions were examined for purity on a 15% SDS-PAGE.

Mammalian Cell Intoxication Assay

Cell cycle arrest after typhoid toxin intoxication was examined by flowcytometry using previously described methods (Spano et al., 2008, CellHost Microbe 3:30-38). Briefly, after treatment with 3×FLAG-taggedtyphoid toxin or bacterial infection for different times (as indicated),cells were trypsinized, harvested, washed, and fixed overnight at −20°C. in ˜70% ethanol/PBS. Fixed cells were washed with PBS and resuspendedin 500 μl of PBS containing 50 μg/m1propidium iodide (PI), 0.1 mg/mlRNase A, and 0.05% Triton X-100. After incubation for 40 min at 37° C.,cells were washed with PBS, resuspended in 500 μl PBS, filtered, andanalyzed by a flow cytometry. DNA contents of cells were determinedusing Flowjo (Treestar).

Light-Scattering Size Exclusion Chromatography and Amino AcidComposition Analysis

Light-Scattering Size Exclusion Chromatography (SEC-LS) and amino acidcomposition analysis were carried out at the Keck Biotechnology ResourceLaboratory at the Yale University School of Medicine. For SEC-LSanalysis, the toxin was purified in 50 mM Hepes buffer containing 150 mMNaCl and run on a Superdex 200 column equipped with a light scatteringdetector using a same Hepes buffer. For amino acid composition analysis,typhoid toxin was resolved on a 15% SDS-PAGE gel, stained with Coomassiebrilliant blue, and the three individual bands were excised and used foramino acid composition analysis.

Mouse Intoxication Experiments

All animal experiments were conducted according to protocols approved byan Institutional Animal Care and Use Committee. Groups of C57BL/6 micewere intravenously injected with 100 μl solutions containing either TBSbuffer alone or 10 μg of each of the purified holotoxin preparations(endotoxin free). His-tagged wild type, PltA catalytic mutant (PltAE133A), CdtB catalytic mutant (CdtB H160Q), double catalytic mutant(PltA E133A CdtB H160Q), and PltB S35A mutant holotoxins were purifiedas described elsewhere herein. Changes in behavior, weight, andtemperature of the toxin-injected mice as well as their survival wereclosely monitored during the duration of the experiment.

Peripheral Blood Leukocyte Preparation, Immunostaining, and FlowCytometry Analysis

Peripheral blood samples of typhoid toxin treated and control mice werecollected into heparinized tubes, incubated with 1 ml ACK lysis buffer(BioWhittaker) (to remove red blood cells), incubated for 5 min on ice,washed with 2 ml PBS, and centrifuged to collect peripheral bloodleukocytes (PBLs). After a repetition of the red blood cell removalstep, PBLs were used for immunostaining as described elsewhere herein.After washing, the cells were immediately incubated for 30 min on icewith 100 μl of anti-mouse Ly-6G (Gr-1) antibody conjugated with FITC(eBioscience). The cells were then washed with 2 ml FACS buffer (PBS,0.16% BSA), resuspended in 100 μl FACS fixation buffer (PBS, 1%paraformaldehyde, 1% FCS), and used for flow cytometric analyses on aFACSCalibur flow cytometer (BD Biosciences). Alternatively, bloodsamples collected by heart puncture 4 days after toxin treatment wereanalyzed in a Hemavet 950FS hematology analyszer (Drew Scientific).

Identification of Typhoid Toxin Interacting, Biotin-Labeled Host CellSurface Proteins

Cultured cells (Henle, Jurkat, Ramos, or THP1 at ˜50% confluency) werewashed with PBS, treated with PBS containing ˜100 μg/ml ofSulfo-NHS-SS-Biotin (Thermo) for 30 min at RT, and subsequently washed 3times with 50 mM Tris-HCl pH8.0/150 mM NaCl buffer to quench and toremove extra biotin reagent. After additional washings (3 times withPBS), cells were resuspended in a lysis buffer containing 50 mMTris-HCl, pH7.4, 150 mM NaCl, 1% NP-40, and lx protease inhibitor,incubated for 20 min on ice, and homogenized by passing a 26-G needle˜20 times.

After removal of cellular debris by centrifugation, the supernatantswere mixed with 10 μg purified FLAG-tagged typhoid toxin and incubatedfor 3 hrs at 4° C. Anti-FLAG antibody-containing agarose beads wereadded, incubated for additional 1 hr at 4° C., washed 5 times with PBS,SDS-PAGE sample buffer was added, boiled, and run on SDS-PAGE. The gelswere transferred to nitrocellulose membranes, blocked with TBScontaining 5% BSA, incubated overnight at 4° C. with Streptavidin-HRP(1:5000) in TBS/1% BSA, washed with TBST, and developed with ECLsubstrates (Pierce).

To identify the typhoid toxin-interacting proteins by LC-MS/MS,equivalently obtained samples were run in parallel, stained withCoomassie blue, and gel regions corresponding to the molecular weight oftyphoid-toxin interacting proteins identified by western blot analysiswere excised and processed for LC-MS/MS using previously describedmethods (Liu et al., 2012, PLoS pathogens 8:e1002562). Briefly, gelslices were destained in destaining buffer (50 mM NH₄HCO₃, 50%Acetonitrile (ACN)), and dehydrated with ACN. Disulfide bonds werereduced by incubating the samples with NH₄HCO₃ containing 10 mM DTT andalkylated by incubating them with 55 mM 2-iodoacetamide in 100 mM H₄HCO₃buffer for 20 min at RT. Gel pieces were dehydrated, trypsin-digestedovernight, extracted, run on an LTQ-Velos Mass Spectrometer, and spectraanalyzed with Mascot (Matrixscience). As negative controls, equivalentlyprocessed samples obtained with two irrelevant baits (GST-3×Flag andInvC-3×Flag) were used.

Oregon Green 488 Typhoid Toxin Labeling

Purified wild type and PltBS35A mutant typhoid toxin preparations werefluorescently labeled with Oregon Green (OG)-488 dye (Invitrogen)according to the vendor's recommendation. OG-488 dye has a succinimidylester moiety that reacts with primary amines of proteins to form stabledye-protein conjugates. Purified toxin preparations (1 mg/ml) wereincubated with reactive dye in 500 μl of 100 mM bicarbonate buffer for 1hr at RT, and applied to a size exclusion chromatography column providedby the vendor to separate the dye-protein conjugates from free dye.Degree of labeling was determined by measuring the absorbance of theconjugate solution at 280 nm and 496 mm, which yielded comparable toxinlabeling for both toxin preparations (4.4:1 and 4.36:1 dye/holotoxinratios for wild type and PltB S35A mutant toxin, respectively). Apredicted extinction coefficient of 191,400 M⁻¹ cm⁻¹ was used tocalculate the dye/toxin ratio.

Typhoid Toxin Binding Assay

Cells were harvested by trypsinization, washed with HBSS, resuspended in100 μl HBSS containing 0.2 μg of Oregon Green-488 (Invitrogen)-labeledpurified wild type or mutant toxin preparations. Cells were incubated inthe presence of the labeled toxin preparations for 30 min at roomtemperature, washed with PBS, resuspended in 100 μl PBS containing 1%paraformaldehyde, and analyzed by flow cytometry. When indicated,Henle-407 cells were treated for 2 hrs with 10 μl deglycosidase mix(NEB) in 2 ml HBSS prior to processing for toxin-binding assays asdescribed elsewhere herein.

Generation of PODXL-Depleted Cell Lines

RNA interference vector pSUPER-H1 (Oligoengine) was used to generate aplasmid expressing an shRNA construct targeted to podxl. Oligosincluding a target region for podx

(SEQ ID NO: 1) 5′-GATCCCCGGACAAATGGGATGAACTATTCAAGAGATAGTTCATCCCATTTGTCCTTTTTC and (SEQ ID NO: 2)5′-TCGAGAAAAAGGACAAATGGGATGAACTATCTCTTGAATAGT TCATCCCATTTGTCCGGGwere annealed to form double-stranded DNA and cloned into the BgIII andHindIII sites of pSUPER-H1 vector. Henle-407 cells were transfected withthis plasmid using Lipofectamine 2000 (Invitrogen) andpuromycin-resistant stable-transfected cell lines were screened forPODXL expression by real time-PCR using a podx1-specific primer set. Theprimer sequences were as follows:

(sense; SEQ ID NO: 3) 5′-ACCGGGGACTACAACCCTG and (antisense; SEQ ID NO: 4) 5′-TGTGGTGTTAGGTTTAGCTGTG for podxl and(sense; SEQ ID NO: 5) 5′-GATTACTGCTCTGGCTCCTAGC and(antisense; SEQ ID NO: 6) 5′-GACTCATCGTACTCCTGCTTGC for β-actin.

Glycan Array Analysis

OG488-labeled wild type and PltB S35A mutant holotoxins were diluted to180 μg/ml or 20 μg/ml and an aliquot (70 μl) was applied to separatemicroarray slides (version 5.1) at the Consortium for FunctionalGlycomics Protein-Glycan Interaction Core, at Emory University. The dataare reported as average relative fluorescence units of four of sixreplicates (after removal of the highest and lowest values) for eachglycan represented on the array. Glycans showing typhoid toxin bindingactivity (listed in FIG. 20) were selected considering a cut off valuethat was larger or equal to than 1% of the values obtained with theglycan showing the highest binding activity. Glycans showing a variationcoefficient higher than 30% were eliminated from this group. In additionsome specific glycans were eliminated from the group because they arephysiologically irrelevant (glycan #509) or showed non-specific binding(glycans #335, 336, 523).

Surface Plasmon Resonance

Surface plasmon resonance analysis was carried out at the Keck

Biotechnology Resource Laboratory at the Yale University School ofMedicine using a BiaCore biosensor. Briefly, 50 μg/ml anti-M2 Flagantibody was immobilized on the surface of a chip by amine coupling.Purified wild type or PltB S35A (both FLAG tagged on the C-terminus ofCdtB) mutant toxin preparations were applied to the chip followed byapplication of Ganglisoside GD2 glycan (Elicityl) at variousconcentrations.

Crystallization

The purification of 6×His-tagged typhoid toxin used for crystallizationis described elsewhere herein. Initial spare matrix crystallizationtrials of full-length holotoxin protein preparations (2 mg/ml) werecarried out at the Yale University School of Medicine Structural BiologyCore facility. After crystal optimization trials, full length typhoidtoxin (4.5 mg/ml) crystals grew in ˜3 weeks at room temperature usingthe hanging-drop vapour-diffusion method in a mix of 1 μl of proteinwith 1 μl of reservoir solution consisting of 1.6 M sodium formate and0.1 M sodium acetate, pH 4.5.

X-Ray Data Collection and Structure Determination

X-ray data were collected to 2.4A at the wavelength of 1.5418 Å on aRigaku Homelab system at the Yale University Chemical and BiophysicalInstrumentation Center (CBIC). Data were integrated and scaled using theHKL-2000 package using previously described methods (Otwinowski andMinor, 1997, Methods Enzymol. 276:307-326). Further processing wasperformed with programs from the CCP4 suite using previously describedmethods (Project, 1994, Acta Crystallogr. D 50:760-763). The holotoxinstructure was determined by molecular replacement using PHASER8 with theatomic coordinates of Chain B of H. ducreyi CDT (PDB ID, 15R4; Nesié etal., 2004, Nature 429:429-433), Chain A of pertussis toxin (PDB ID,1PRT; Stein et al., 1994, Nat. Struct. Biol. 1:591-596) and Subtilasecytotoxin B-subunit (PDB ID, 3DWP; Byres et al., 2008, Nature456:2126-2132) as the initial search model. The atomic coordinates havebeen deposited in the RCSB Protein Data Bank (entry number 4KSL).

To complete the model, manual building was carried out in COOT usingpreviously described methods (Emsley and Cowtan, 2004, Acta Crystallogr.D 60:2126-2132). Figures were prepared using PyMol using previouslydescribed methods (Delano, 2002, The PyMOL Molecular Graphics System;www.pymol.org). The structure refinement was done by PHENIX usingpreviously described methods (Adams et al., 2010, Acta Crystallogr. D66:213-221). The data collection and refinement statistics aresummarized in FIG. 21.

Molecular Docking

Molecular docking of Neu5Ac onto PltB was carried out with AutoDock Vinausing previously described methods (Trott and Olson, 2010, J.Computational Chem. 31:455-461). Based on the available structural andfunctional information on pertussis and subtilase toxins as well asfunctional data indicating the importance of PltB Ser35 in sugar binding(FIG. 4), the binding of Neu5Ac was modeled onto a pocket surroundingPltB Ser35 and consider several amino acid residues (Tyr33, Tyr34, Ser35and Lys59) as flexible. The calculation yielded 20 possible models, ofwhich the one with the highest ranking was selected as the most likely.

Fluorescence Microscopy

Fluorescence microscopy analysis of typhoid toxin in S. typhi infectedcells was carried out using previously described methods (Spano et al.,2008, Cell Host Microbe 3:30-38). Briefly, Henle-407 cells were seededon coverslips placed within 24-well plates and cultured overnight.Cultured cells were infected with different strains of S. typhiexpressing FLAG-epitope tagged typhoid toxin with a multiplicity ofinfection of 20 for 1 hr. Infected cells were washed, treated for 1 hrwith 100 μg/ml gentamicin to kill extracellular bacteria, washed again,and incubated for 24 hr in a cell culture medium containing 10 μg/m1gentamicin. Infected cells were washed with PBS, fixed with 4%paraformaldehyde for 10 min at RT, washed, and incubated for 30 min atRT with PBS containing 50 mM NH₄Cl, 0.2% Triton X-100 and 3% BSA.

Cells were then incubated for 30 min at RT with a primary antibodymixture of mouse anti-Flag M2 (Sigma; 1:4000) and rabbit anti-S. typhi(Difco; 1:4000) in PBS containing 3% BSA, washed, incubated for 30 minat RT with a secondary antibody mixture of Alexa-488 anti-mouse (1:2000)and Alexa-594 anti-rabbit (1:2000) in PBS containing 3% BSA. Cells werewashed, stained with DAPI (1:10,000), washed again, mounted, and viewedusing a 100× objective on a fluorescence microscope (Nikon TE2000).Puncta intensities of images were analyzed using ImageJ software usingmethods previously described (Spano et al., 2011, Proc. Natl. Acad. Sci.USA 108:18418-18432).

Statistics

The two-tailed student T-test was performed to determine the statisticalsignificance of experimental changes from control values. A p value ofless than 0.05 was considered significant.

Sequences

CdtB; NP_456275.1

(SEQ ID NO: 7) MKKPVFFLLTMIICSYISFACANISDYKVMTWNLQGSSASTESKWNVNVRQLLSGTAGVDILMVQEAGAVPTSAVPTGRHIQPFGVGIPIDEYTWNLGTTSRQDIRYIYHSAIDVGARRVNLAIVSRQRADNVYVLRPTTVASRPVIGIGLGNDVFLTAHALASGGPDAAAIVRVTINFFRQPQMRHLSWFLAGDFNRSPDRLENDLMTEHLERVVAVLAPTEPTQIGGGILDYGVIVDRAPYSQRVEALRNPQLASDHYPVAFLARSC

PltA; NP_456278

(SEQ ID NO: 8) MKKLIFLTLSIVSFNNYAVDFVYRVDSTPPDVIFRDGFSLLGYNRNFQQFISGRSCSGGSSDSRYIATTSSVNQTYAIARAYYSRSTFKGNLYRYQIRADNNFYSLLPSITYLETQGGHFNAYEKTMMRLQREYVSTLSILPENIQKAVALVYDSATGLVKDGVSTMNASYLGLSTTSNPGVIPFLPEPQTYTQQRIDAFGPLISSCFSIGSVCHSHRGQRADVYNMSFYDARPV IELILSK

PltB; NP_456279.1

(SEQ ID NO: 9) MYMSKYVPVYTLLILIYSFNASAEWTGDNTNAYYSDEVISELHVGQIDTSPYFCIKTVKANGSGTPVVACAVSKQSIWAPSFKELLDQARYFYSTGQSVRIHVQKNIWTYPLFVNTFSANALVGLSSCSATQCFGPK

The results of the experiments are now described.

A₂B₅ Organization of Typhoid Toxin

To examine the potential role of typhoid toxin in the acute phase oftyphoid fever, a protocol to obtain a highly purified preparation ofactive holotoxin was developed (FIGS. 1A-1C). It was observed thatsystemic administration of typhoid toxin into mice caused many of thesymptoms observed during the acute phase of typhoid fever. Despite thelack of fever (FIG. 5), the mice appeared lethargic showing clear signsof stupor and malaise. Furthermore, the mice lost weight (FIG. 1D) andeventually died (FIG. 1E). The toxin-injected animals also showed asignificant reduction in the number of circulating immune cells,resulting in the almost complete depletion of circulating neutrophils(FIGS. 1F-1G), a phenotype often observed during the acute phase oftyphoid fever. Consistent with this observation, typhoid toxin was ableto intoxicate a broad range of cells in vitro, including severalepithelial and immune cells (FIG. 6). Although symptoms were observed inanimals inoculated with a toxin carrying a catalytic mutant of PltA(FIG. 1D), no detectable symptoms were observed when animals wereinoculated with equally purified preparations of typhoid toxin carryinga catalytic mutant of its CdtB subunit (FIGS. 1D-1G). Although notwishing to be bound by any particular theory, when taken together, theseresults indicate that typhoid toxin, through its CdtB subunit, maycontribute to the acute symptomatology observed during typhoid fever.

To gain further insight into the mechanism of action of typhoid toxin,its cellular receptor or receptors were identified. A highly purifiedbiologically active typhoid toxin preparation was used to affinitypurify biotin-labeled host cell surface interacting proteins. It wasobserved by LC-MS/MS analyses of interacting proteins that in humanHenle-407 epithelial cells, typhoid toxin interacts withPodocalyxin-like protein 1 (PODXL) (FIGS. 2A-2B). PODXL is a member ofthe CD34 sialomucin protein family, which localizes to the apical sideof epithelial cells and is also expressed in vascular endothelial cells(Ue et al., 2007, Mol. Biol. Cell. 18:1710-1722). Consistent with itspotential role as a toxin receptor, shRNA-mediated depletion of PODXLresulted in a significant reduction in toxin binding (FIG. 2C and FIG.7) and toxin-mediated cell cycle arrest (FIG. 2D).

Because it was observed that in addition to the intoxication ofepithelial cells, typhoid toxin is capable of intoxicating a broad rangeof cells (FIG. 6), the interaction of typhoid toxin with surfaceproteins of other cell lines was examined by affinity purification andLC-MS/MS analyses. It was found that in macrophages, as well as in T andB cells, typhoid toxin interacts with receptor tyrosine phosphatase C,also known as CD45 (FIG. 8), which is ubiquitously expressed inhematopoietic cells other than erythrocytes and platelets (Hermiston etal., 2009, Immunol. Rev. 228:288-311). Although not wishing to be boundby any particular theory, these results suggest that typhoid toxin mayengage different receptors in different cells.

The identified typhoid toxin-interacting proteins, however, are allheavily glycosylated. It was hypothesized that typhoid toxin mayinteract with these different surface proteins through commoncarbohydrate moieties. This hypothesis was tested by examining typhoidtoxin binding to cells that had been pre-treated with a mixture ofglycosidases. It was observed that removal of surface glycanssignificantly reduced typhoid toxin binding (FIG. 2E). Furthermore, acell line lacking all complex and hybrid N-glycans on glycoproteins dueto a mutation in N-acetylglucosaminyltransferase I (Kumar et al., 1990,Proc. Natl. Acad. Sci. USA 87:9948-9952; Stanley et al., 1975, Proc.Natl. Acad. Sci. USA 3323-3327) was more resistant to typhoid toxin thanits parent wild type cell line (FIGS. 2F, 2G, and FIG. 9). Although notwishing to be bound by any particular theory, these results indicatethat a sugar moiety(s) on surface glycoproteins serves as a receptor fortyphoid toxin.

The nature of the glycan moiety on host cells that is recognized bytyphoid toxin was identified. 610 glycans arrayed on a solid surface(Song et al., 2011, Nat. Methods 8:2085-2090) were probed for binding tobiologically active, highly purified, fluorescently labeled typhoidtoxin. This analysis revealed a complex binding pattern involving 4 mainglycan groups (FIGS. 2H, 20, and 21). The first group, which is mostcommonly present in complex N-linked glycans, is represented bysialylated tri- or bi-antennary glycans (e. g. glycans #461, #483, and#482) with one or all of the branches terminally sialyated (note: glycannumbers correspond to the nomenclature used by the Consortium forFunctional Glycomics, www.functionalglycomics.org). This group includesglycans with both Neu5Aca2-3 (e. g. #483) and Neu5Aca2-6 (e. g. #482)terminal linkages. However, although not wishing to be bound by anyparticular theory, typhoid toxin likely binds preferentially toNeu5Aca2-3 terminal linkages since glycan #483 showed stronger bindingthan glycan #482, which only differ in their terminal linkages (FIGS.2H, 20, and 21). Furthermore, although not wishing to be bound by anyparticular theory, typhoid toxin likely preferentially bindstri-antennary over bi-antennary compounds, since glycan #461 exhibitedthe highest binding affinity. In addition, although not wishing to bebound by any particular theory, the toxin may also bind preferentiallythe type 2-N-acetyllactosamine unit (Ga1β1-4G1cNAc), present in glycan#461, over type 1-N-acetyllactosamine unit (Ga1β1-3G1cNAc), present inthe very similar tri-antennary N-glycan #474, which showed lower bindingaffinity (FIGS. 2H, 20, and 21).

The second group consists of non-sialylated tri- or bi-antennary glycansalso commonly found in complex N-linked glycans. Overall this groupexhibited lower binding affinity than sialylated glycans. Although notwishing to be bound by any particular theory, this result suggests apreference of typhoid toxin for the terminal sialic acid modification.

The third group consists of glycans commonly found in glycolipids,mostly as gangliosides. Six glycans were identified within this group(out of 75 present in the array) that showed toxin binding with variousaffinities. Although not wishing to be bound by any particular theory,the Neu5Acα 2-8 Neu5Ac disialoside group, present in ganglioside GD2gangliosides (Yu et al., 2011, J. Oleo Sci. 60:537-544), (glycan #228),is likely preferred by the toxin since ganglioside GM3 (glycan #263)containing Neu5Ac monosialoside did not show toxin binding. Although notwishing to be bound by any particular theory, these results suggest thatin certain cells, typhoid toxin may also be able to use glycolipids as areceptor. Consistent with this hypothesis, typhoid toxin was able tointoxicate an N-acetylglucosaminyltransferase I-deficient cell linealthough only when applied at high concentrations (FIG. 10).

The fourth group is defined by glycans commonly found on O-glycans.Among them is glycan #243, which shares the canonical structure of mucintype O-GalNAcylated glycan. Other glycans in this group share thecanonical structure of O-GlcNAcylated glycans. The significance of thisgroup for toxin-binding in vivo is unclear since they bind with lowaffinity and, unlike the other groups of glycans, they are mostly foundwithin the nucleus and not on surface glycoproteins (Stein et al., 1994,Nature Struc. Biol. 1:591-596). Overall, typhoid toxin exhibitsbroad-binding specificity similar to that observed for pertussis toxin(Millen et al., 2010, Biochemistry 49:5954-5967), which also targets alarge variety of cells, but significantly different from the muchnarrower specificity exhibited by other ABS toxins, such as subtilasecytotoxin, which specifically recognizes glycans terminating inN-glycolylneuraminic acid (Neu5Gc) (Byres et al., 2008, Nature456:648-652). However, unlike pertussis toxin, in which each of theheteromeric B subunits contributes diversity to the binding specificity(Millen et al., 2010, Biochemistry 49:5954-5967), typhoid toxin achievesthis broad binding specificity with a single polypeptide, PltB, whichforms its homomeric B subunit. Taken together, these results support thehypothesis that typhoid toxin can use as receptors a broad range ofglycans preferentially on surface glycoproteins but also, although lessefficiently, on surface glycolipids, providing a mechanistic explanationfor its broad cell target specificity.

To uncover the organization of typhoid toxin, its crystal structure wassolved to 2.4 Å resolution. The structure showed a complex of 5 PltBmolecules and one molecule each of PltA and CdtB (FIG. 3A and Table 1),which is consistent with the stoichiometry observed by size exclusionchromatography combined with dynamic light scattering and quantitativeamino acid composition analysis (FIG. 11).

TABLE 1 Statistics of Data Collection and Refinement Data typhoid toxinIntegrate package HKL2000 Space Group C222₁ Unit Cell 78.386, 261.076,109.896 (a, b, c in Å, β in degrees) 90, 90, 90 Wavelength (Å) 1.5418Resolution (Å) 31.91-2.393 (2.479-2.393) R_(merge) (%) 9.7 (72.4)I/sigma 12.81 (2.62) Completeness (%) 97.14 (93.03) No. of reflections284503 No. of unique reflections 43611 Redundancy 6.5 (5.8) Wilson Bfactor (Å) 41.49 R/R_(free) (%) 0.2121 (0.3119)/0.2536 (0.3386) No. ofatoms Overall 8526 Macromolecules 8102 Glycerol 48 Water 376 Average Bvalue (Å) Overall 48.70 Macromolecules 49.00 Solvent 42.50 R.m.s.deviations Bond (Å) 0.004 Angle (°) 0.86 Ramachandran plot statistics(%) Most favorable 89.9 Additionally allowed 9.0 Generously allowed 1.1Disallowed 0.0 Values in parentheses are for the highest resolutionshell.

These results indicate an unprecedented A₂B₅ organization for typhoidtoxin, which is in contrast to other known AB₅ toxins that have only oneA subunit (Beddoe et al., 2010, Trends Biochem. Sci. 35:411-418; Merritand Hol, 1995, Curr. Opin. Struct. Biol. 5:165-171). The pyramid shapedcomplex has a height of ˜90 Å and a maximum width of ˜60 Å (FIG. 3A),with CdtB located at the vertex, connected by PltA to a pentameric discat the base of the pyramid made of 5 monomers of PltB (FIGS. 3A-3B). Thetandem linear arrangement of the enzymatic subunits PltA and CdtBdictates that there are no interactions between CdtB and PltB. Aspredicted by the amino acid sequence similarities, the PltA and CdtBsubunits exhibit a very similar structure to the pertussis toxin S1 (andother ADP ribosyl transferases; Stein et al., 1994, Structure 2:45-57)and to the CdtB subunit of Cytolethal distending toxin (Nesié et al.,2004, Nature 429:429-433).

PltA aligns very well to the pertussis toxin S1 domain with aroot-mean-squared deviation (rmsd) of 2.168 Å over 140 Cα atoms (with31% sequence identity) (FIG. 12). The positions of the conservedcatalytic residues (Glu133 in typhoid toxin and Glu129 in pertussistoxin S1), as well as the disulfide bonds (Cys56-Cys207 in typhoid toxinand Cys41-Cys201 in pertussis toxin S1) overlap almost completely (FIG.12). The latter indicates that, similarly to the pertussis toxin S1subunit (Stein et al., 1994, Structure 2:45-57; Locht et al., 2011, FEBSJ. 278:4668-4682), PltA would have to be reduced to allow the access ofNAD and its putative substrates to the active site, and consequently, areducing activating step must be necessary prior to contacting its hostcell target(s). Typhoid toxin CdtB aligns very well to the Haemophilusducreyi CdtB with an rmsd of 0.947 Å over 206 Cα atoms (with 52%sequence identity) (FIG. 13). The positions of the conserved catalyticresidues in Typhoid toxin's CdtB overlap almost completely with those ofits homolog in H. ducreyi.

Similar to the B subunits of other AB₅ toxins (Beddoe et al., 2010,Trends Biochem Sci. 35:411-418; Byres et al., 2008, Nature 456:648-652),the PltB oligomer is arranged as a pentamer with a central channel thatis lined by 5 helixes (FIG. 3B). As predicted from its amino acidsequence similarity and consistent with the data presented above, thePltB protomer shows a typical oligosaccharide-binding fold on the sideof the pentamer (FIGS. 3C and 14), a location similar to that of toxinsthat preferentially bind glycoproteins but different from those thatpreferentially bind glycolipids, which have the sugar-binding pockets onthe membrane-proximal face of the toxin (Beddoe et al., 2010, TrendsBiochem Sci. 35:411-418). Therefore, these findings are consistent withthe observation that typhoid toxin preferentially binds glycans presenton surface glycoproteins over those present on glycolipids (FIGS. 20 and21).

The monomer of typhoid holotoxin PltB aligns very well with SubB, thesubtilase cytotoxin B subunit, with an rmsd of ˜0.5 Å over 97 Cα atoms(with 50% sequence identity) (FIG. 14). Of note, the positions of theconserved putative sugar-binding residue Ser35 overlap very well,although in SubB Ser35 is located within a β-strand while in PltB isplaced within a loop (FIGS. 3C and 14). The predicted sugar-bindingpocket in PltB is not as deep and appears more extended than in SubB,which also differs in surface charge distribution (FIG. 3C). Althoughnot wishing to be bound by any particular theory, these differences mayaccount for the significantly different binding specificities exhibitedby these two toxins (Byres et al., 2008, Nature 456:648-652). PltB wasalso compared to the pertussis toxin S2 B subunit (Stein et al., 1994,Nat. Struct. Biol. 1:591-596). Although their overall amino sequencesimilarity is low, the structures can be aligned very well around theirsugar-binding domains with an rmsd of 1.752 Å over 80 Cα atoms (FIG.14).

Residues in pertussis toxin S2 involved in sugar binding (Tyr102,Ser104, Arg125) are well conserved in PltB (Tyr33, Ser35, Lys59) (FIG.3D), and the charge distribution and architecture of the sugar-bindingpockets are similar (FIG. 3C). This is consistent with the observationthat, despite overall less conservation, these two B subunits sharesugar-binding specificity. For example, several of the glycans that bindtyphoid toxin possess an Neu5Ac moiety at their terminal end, adeterminant that also binds pertussis toxin (Millen et al., 2010,Biochemistry 49:5954-5967). Structural modeling of Neu5Ac bound to PltBpredicts almost identical interaction to those observed in the atomicstructure of pertussis toxin bound to Neu5Ac (FIGS. 3C-3D and FIG. 15).

Consistent with the structural predictions, a mutation in thesugar-binding pocket of PltB (PltB Ser35A) abrogated the ability oftyphoid toxin to bind glycans in glycoarray (FIGS. 4A and 22) andsurface plasmon resonance assays (FIG. 16), and the ability of the toxinto bind (FIG. 4B) and intoxicate (FIG. 4C) cultured cells or to causesymptoms when systemically applied to mice (FIG. 4D). These results arealso consistent with the explanation that there is a singlecarbohydrate-binding domain in typhoid toxin since the mutant abrogatedbinding to all carbohydrates. Surface plasmon resonance assays alsoindicated that on average at least for the glycan tested (GD2), eachPltB pentamer binds 2.5 sugar molecules with an affinity of ˜1.2 mM(FIG. 16). However, the glycoarray analysis predicts that the bindingaffinity is likely to be higher for other more complex glycans.

The interaction of PltA with the PltB oligomer occurs largely throughits carboxy terminus, which buries 1,657 Å² and has a ΔiG of −17.7kcal/mol. A critical element in this interaction is a short helix at thecarboxyterminal end of PltA, which inserts into the hydrophobic lumen ofthe PltB channel (FIGS. 3B and 17) and stabilizes the complex bycritical interactions mediated by Pro234, Va1235, Ile236, Leu238,Ile239, and Leu240 in PltA.

The most salient feature of the interface between PltA and CdtB is adisulfide bond between Cys214 of PltA and Cys269 of CdtB (FIG. 3E). Therest of the interface between the two A subunits is unremarkable,burying only 950 Å² with a ΔiG of only −5.2 kcal/mol. Although notwishing to be bound by any particular theory, this observation suggeststhat the disulfide bound is essential for the integrity of theholotoxin. Consistent with this hypothesis, subjecting the typhoid toxinto reducing conditions resulted in the dissociation of CdtB from thePltA-PltB complex (FIG. 4E). Furthermore, introduction of a mutation inCys269 of CdtB destabilized the holotoxin complex in vitro (FIG. 4F),and resulted in a loss of CdtB-dependent toxicity in S. typhi-infectedcells (FIGS. 4G and 18). This mutation also prevented the assembly ofCdtB into its vesicle carrier intermediates that can be visualized asfluorescent puncta in S. typhi-infected cells (FIGS. 4H-4I), despite thefact that the mutant was expressed at equivalent levels to those of wildtype (FIG. 19). These results support the hypothesis that the CdtBCys269/PltACys214 disulfide bond is critical for the assembly of typhoidtoxin holotoxin in the periplasm of the bacteria.

Even though the sequence conservation between typhoid toxin's CdtB andthat of its cytolethal distending toxin homologs is very high, theCys269 is unique to Typhoid toxin's CdtB (FIG. 4J). Likewise, althoughall close homologs of PltA including ArtA, a closely related mono ADPribosyl transferase from S. typhimurium (Saitoh et al., 2005,Microbiology 151:3089-3096), have only two Cys that form anintramolecular disulfide bond, PltA is unique in that it has a third Cys(Cys214) to pair with S. typhi's CdtB (FIG. 4J). Therefore typhoidtoxin's CdtB has been specifically adapted for its tethering to thePltA/PltB complex by the evolution of uniquely positioned Cys residuesin both PltA and CdtB. The covalent linkage of CdtB to the PltA/PltBcomplex by a disulfide bond thus ensures that CdtB and PltA aresimultaneously delivered to the same target cell. Furthermore, thisarrangement would ensure that, after endocytosis and retrogradetransport to its place of translocation (most likely the endoplasmicreticulum), PltA and CdtB would be freed from one another upon reductionof the disulfide bond by local reductases, thus allowing them to becomecompetent for translocation to the cytosol and delivery to their placeof action.

These results provide unique insight into typhoid toxin, a criticalvirulence factor of S. typhi, revealing unprecedented features for abacterial exotoxin and providing significant information on thepathogenesis of typhoid fever. The potential implication of typhoidtoxin in the development of symptoms during the acute, life threateningphase of typhoid fever, combined with its unique architecture, can beuseful for the development of novel, potentially life-saving therapeuticinterventions against this disease.

Immune Response to Typhoid Toxin

To test the ability of typhoid toxin to stimulate the production ofneutralizing antibodies, mice were immunized with a purified preparationof inactivated typhoid toxin (“toxoid”). The typhoid toxin toxoid wasgenerated by introducing mutations in the catalytic sites of its twoenzymatic subunits, PltA (PltAE133A) and CdtB (CdtBH160Q). Animalsimmunized with the toxin mounted a strong immune response to the toxingenerating toxin neutralizing antibodies (FIG. 23). Importantly,antibody responses were generated even in the absence of animmunological adjuvant, suggesting that the toxin itself may haveadjuvant activity. In contrast, animals inoculated with an otherwiseidentical typhoid toxin toxoid preparation but carrying a mutation inthe glycan-binding site of its PltB “B” subunit (PltBS35A) was only ableto stimulate an immune response when administered with an immunologicaladjuvant (e.g., Alumn). Although not wishing to be bound by anyparticular theory, these results are consistent with the explanationthat Typhoid toxin has immune adjuvant activity and that such activityis dependent on the toxin's ability to bind glycans since aglycan-defective mutant did not stimulate a strong immune response inthe absence of an adjuvant (FIG. 23). These observations have profoundimplications for the potential design of a typhoid fever vaccine.Current vaccines against typhoid fever are composed of the so called Viantigen, a surface polysaccharide of Salmonella Typhi. To render itsufficiently immunogenic, the polysaccharide is conjugated to a proteincarrier. In addition, an immunological adjuvant is required to stimulateimmune responses to the polysaccharide-protein conjugate. The resultsdescribed herein support the conclusion that a Typhoid toxin toxoidcould serve as 1) a potent immunogen to stimulate anti-toxin immunity;2) a protein carrier (to stimulate immune responses to polysaccharideantigens such as, by way of non-limiting example, the Vi antigen); and3) as an immunological adjuvant to stimulate an immune response.

As shown herein, patients convalescent of typhoid fever mount a strongserum immune response to typhoid toxin. Due to the lack of specificsymptomatology diagnosing typhoid fever remains a challenge (Bhutta,2006, BMJ 333:78-82; Parry et al., 2011, Expert Rev Anti Infect Ther9:711-725). A positive blood culture test remains the gold standard, butsince the bacterial load in the blood is so low, this test is oftennegative even in positive cases of typhoid fever. Thus, there is anurgent need for a practical, rapid test to diagnose typhoid fever.Although there are a number of serological tests such as the Widal test,their specificity and sensitivity are extremely poor. Typhoid toxin isan ideal antigen to diagnose typhoid fever since it is the only antigenknown that is specific for the only two Salmonella enterica serovarsthat can cause typhoid fever: Salmonella Typhi and Salmonella Paratyphi.Patients convalescent of typhoid fever had a high serum antibody levelsagainst typhoid toxin. Importantly, antibody levels were high uponadmission to the Hospital, and remain high for several weeks (FIG. 24).In contrast, control patients with no history of typhoid fever had noserum antibodies to the toxin. These results indicate that typhoid toxincan provide the bases for the development of a diagnostic test fortyphoid fever.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A vaccine comprising at least one polypeptideselected from the group consisting of: a. PltA, or a PltA mutant; b.PltB, or a PltB mutant; and c. CdtB or a CdtB mutant.
 2. The vaccine ofclaim 1, wherein the PltA mutant is at least one selected from the groupconsisting of PltA E133X, relative to SEQ ID NO: 8 and PltA E133A,relative to SEQ ID NO:
 8. 3. (canceled)
 4. The vaccine of claim 1,wherein the PltB mutant is at least one selected from the groupconsisting of PltB S35X, relative to SEQ ID NO: 9 and PltB 535A,relative to SEQ ID NO:
 9. 5. (canceled)
 6. The vaccine of claim 1,wherein the CdtB mutant is at least one selected from the groupconsisting of CdtB H160X, relative to SEQ ID NO: 7, CdtB H160Q, relativeto SEQ ID NO: 7, CdtB H160Q, relative to SEQ ID NO: 7, CdtB R119X,relative to SEQ ID NO: 7, CdtB H259X, relative to SEQ ID NO: 7, CdtBΔCys269, relative to SEQ ID NO: 7, CdtB C269X, relative to SEQ ID NO: 7.7. The vaccine of claim 1, wherein the vaccine comprises a bacterium. 8.(canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. The vaccineof claim 1, wherein the PltA mutant comprises any mutation in PltA thatdisrupts its enzymatic activity.
 13. The vaccine of claim 1, wherein theCdtB mutant comprises any mutation in CdtB that disrupts its enzymaticactivity.
 14. A method of immunizing a subject against S. typhi, themethod comprising administering the vaccine of claim 1 to the subject.15. The method of claim 14, wherein the subject is currently infectedwith S. typhi or S. paratyphi and the vaccine induces an immune responseagainst S. typhi or S. paratyphi.
 16. The method of claim 14, whereinthe subject is not currently infected with S. typhi or S. paratyphi andthe vaccine induces an immune response against S. typhi or S. paratyphi.17. A method of treating a subject infected with S. typhi, the methodcomprising administering the vaccine of at least one of claims claim 1to the subject.
 18. The method of claim 17, further comprising theadministration of an antibiotic.
 19. An isolated polypeptide selectedfrom the group consisting of PltA E133X, PltA E133A, PltB S35X, PltBS35A, CdtB 11160X, CdtB H160Q, CdtB R119X, CdtB H259X, CdtB ACys269, andCdtB C269X.
 20. An isolated nucleic acid encoding an amino acid sequenceselected from the group consisting of PltA E133X, PltA E133A, PltB S35X,PltB S35A, CdtB H160X, CdtB H160Q, CdtB R119X, CdtB H259X, CdtB ACys269,and CdtB C269X.
 21. An antibody the specifically binds to at least oneselected from the group consisting of PltA E133X, PltA E133A, PltB S35X,PltB S35A, CdtB H160X, CdtB H160Q, CdtB R119X, CdtB H259X, CdtB ACys269,and CdtB C269X.
 22. A method of treating a subject infected with S.typhi, the method comprising administering to the subject at least oneantibody, wherein the at least one antibody specifically binds to atleast one of PltA, PltA E133X, PltA E133A, PltB, PltB S35X, PltB S35A,CdtB, CdtB 11160X, H160Q, CdtB R119X, CdtB H259X, CdtB C269X and CdtBACys269.
 23. The method of claim 22, further comprising theadministration of an antibiotic.
 24. An inhibitor composition useful fortreating or preventing S. typhi infection, wherein the inhibitorcomposition inhibits the interaction between the S. typhi toxin and theS. typhi toxin receptor.
 25. The inhibitor composition of claim 24,wherein the inhibitor composition specifically binds to the S. typhitoxin.
 26. The inhibitor composition of claim 24, wherein the inhibitorcomposition specifically binds to the S. typhi toxin receptor.
 27. Theinhibitor composition of claim 24, wherein the S. typhi toxin receptoris a glycan.
 28. The inhibitor composition of claim 24, wherein theinhibitor composition is at least one selected from the group consistingof a chemical compound, a protein, a peptide, a peptidomemetic, anantibody, a ribozyme, a small molecule chemical compound, a glycan, anantisense nucleic acid molecule.
 29. The inhibitor composition of claim24, wherein the inhibitor composition comprises at least one glycanlisted in FIG. 20, 21 or
 22. 30. The inhibitor composition of claim 27,wherein the glycan is soluble.
 31. A method of treating a subjectinfected with S. typhi, the method comprising administering to thesubject the inhibitor composition of claim
 24. 32. The method of claim31, further comprising the administration of an antibiotic.
 33. A methodof diagnosing an S. typhi or S. paratyphi infection in a subject in needthereof, the method comprising: a. determining the level of at least oneof PltA, PltB, CdtB in a biological sample of the subject, b. comparingthe level of the at least one of PltA, PltB, CdtB in the biologicalsample with level in a comparator control, and c. diagnosing the subjectwith an infection by S. typhi or S. paratyphi when the level of the atleast one of PltA, PltB, CdtB in the biological sample is elevated whencompared with the level in the comparator control.
 34. The method ofclaim 33, wherein the level of the at least one of PltA, PltB, CdtB inthe biological sample is determined by measuring the level of at leastone of PltA mRNA, PltB mRNA, CdtB mRNA in the biological sample.
 35. Themethod of claim 33, wherein the level of the at least one of PltA, PltB,CdtB in the biological sample is determined by measuring the level of atleast one of PltA polypeptide, PltB polypeptide, CdtB polypeptide in thebiological sample.
 36. The method of claim 33, wherein the comparatorcontrol is at least one selected from the group consisting of: apositive control, a negative control, a historical control, a historicalnorm, or the level of a reference molecule in the biological sample. 37.The method of claim 33, further comprising the step of administering atherapy to the subject to treat the infection.
 38. A method ofdiagnosing an S. typhi or S. paratyphi infection in a subject in needthereof, the method comprising: a. deteiinining the level of antibodythat specifically binds to at least one of PltA, PltB, CdtB in abiological sample of the subject, b. comparing the level of antibodythat specifically binds to the at least one of PltA, PltB, CdtB in thebiological sample with level in a comparator control, and c. diagnosingthe subject with an infection by S. typhi or S. paratyphi when the levelof the antibody that specifically binds to at least one of PltA, PltB,CdtB in the biological sample is elevated when compared with the levelin the comparator control.
 39. The method of claim 38, wherein thecomparator control is at least one selected from the group consistingof: a positive control, a negative control, a historical control, ahistorical norm, or the level of a reference molecule in the biologicalsample.
 40. The method of claim 38, further comprising the step ofadministering a therapy to the subject to treat the infection.